CN114324617B - Method for separating and detecting related impurities in betamethasone raw material - Google Patents
Method for separating and detecting related impurities in betamethasone raw material Download PDFInfo
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- CN114324617B CN114324617B CN202010607425.4A CN202010607425A CN114324617B CN 114324617 B CN114324617 B CN 114324617B CN 202010607425 A CN202010607425 A CN 202010607425A CN 114324617 B CN114324617 B CN 114324617B
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Abstract
The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and measuring related impurities in a betamethasone raw material. The invention adopts high performance liquid chromatography to carry out separation and determination, adopts a chromatographic column to carry out gradient elution by taking octadecylsilane chemically bonded silica as a filler and water, acetonitrile and methanol as mobile phases. Impurities which cannot be effectively separated by the betamethasone EP10.0 related substance analysis method can be well separated from main peaks of the test sample and other impurities possibly existing in the test sample within 37 minutes, and the problem of impurity separation and determination in betamethasone which cannot be solved by the prior art is provided; and the analysis method has high sensitivity, strong specificity, good repeatability and simple and feasible operation.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for separating and detecting related impurities in betamethasone raw materials.
Background
Betamethasone is a drug, and betamethasone has more potent glycometabolism and anti-inflammatory effects than hydrocortisone, 15 times that of hydrocortisone, but sodium retention is more than hundred times that of hydrocortisone, and can be used in substitution therapy with glucocorticoids in primary adrenocortical insufficiency. Also suitable for the low renin low aldosterone syndrome and the orthostatic hypotension caused by the autonomic neuropathy.
Betamethasone dipropionate can be used for treating skin inflammation and pruritus, such as neurodermatitis, contact dermatitis, seborrheic dermatitis, eczema, localized pruritus, discoid lupus erythematosus, etc. And the betamethasone is used as a starting material of betamethasone dipropionate, and the betamethasone content analysis method is researched, so that errors in the detection process, time cost and the like can be effectively reduced.
The betamethasone has EP10.0, USP42S2 and JP17 pharmacopoeia methods at the present stage, wherein the betamethasone EP10.0 related substance analysis method with better separation effect on known impurities and other related impurities is used for analyzing the impurity SM 1r And impurity SM 1b Cannot be effectively separated, and the impurity SM 1d And impurity SM 1q Failure to achieve baseline separation
The invention provides a more optimized analysis method based on the original basic method. Establishes an HPLC method, impurity SM 1r And impurity SM 1b Effectively separate and remove the impurity SM 1d And impurity SM 1q Is more effective than otherThe method for analyzing the substances related to the bentazone EP10.0 is better.
Disclosure of Invention
It is an object of the present invention to provide a method for separating relevant impurities from a betamethasone feedstock, which can effectively separate relevant impurities from 11 in the betamethasone feedstock.
In order to achieve the above purpose, the present invention provides the following solutions:
the method for separating relevant impurities in betamethasone raw materials adopts a chromatographic column which uses octadecylsilane chemically bonded silica as a filler, and adopts a mobile phase A, a mobile phase B and a mobile phase C for gradient elution to separate the relevant impurities; the mobile phase A is water, the mobile phase B is acetonitrile, and the mobile phase C is methanol; the related impurities are one or more of SM1a, SM1b, SM1c, SM1d, SM1e, SM1g, SM1k, SM1l, SM1q, SM1r and 5, 5-dimethyl hydantoin, and the specific structural formula is as follows:
further, the column temperature of the chromatographic column is 28-32 ℃;
specifically, the column temperature of the chromatographic column is 30 ℃;
further, the column gauge is 4.6mm x 250mm,5 μm;
further, the model of the chromatographic column is Shim-Pack VP-ODS;
furthermore, the sample injection amount of the method is 10-30 mu l;
specifically, the method has a sample injection amount of 10 μl;
further, the gradient elution procedure was:
further, the flow rate of the mobile phase is 0.9-1.1 ml/min;
specifically, the mobile phase flow rate was 1.0ml/min.
The second object of the invention is to provide a method for detecting the related impurities in the raw material of betamethasone, which can accurately judge whether the raw material of betamethasone contains the related impurities.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the method for detecting the relevant impurities in the betamethasone raw material comprises the steps of separating the relevant impurities in the betamethasone raw material by the method in the first aim, detecting by a detector, setting the wavelength of the detector to 245-265 nm, comparing a chromatogram of a sample to be detected with a chromatogram of a known reference substance, and judging whether the betamethasone raw material contains the relevant impurities;
further, the detector wavelength is set to 254nm.
The invention further aims to provide a method for determining the related impurities in the betamethasone raw material, which can accurately determine the content of the related impurities in the betamethasone raw material.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method for determining relevant impurities in a betamethasone feedstock, characterized in that the method comprises the specific steps of:
1) Preparing a test sample solution and a reference substance solution: dissolving a sample to be tested in a diluent to obtain a sample solution; dissolving the related impurity reference substance in a diluent to obtain a reference substance solution;
2) Separating: separating relevant impurities in the betamethasone raw material by the method in the first aim;
3) And (3) detection: detecting the presence of the relevant impurity in the raw material of the tadalasone by using the method described in the second aim;
4) And (3) measuring: calculating the content of the related impurities in the sample solution according to the detected chromatogram and the peak area by an external standard method;
specifically, the diluent is methanol and acetonitrile in a volume ratio of 1:1.
The invention has the beneficial effects that:
(1) The invention establishes a stable and reliable experimental method for detecting relevant impurities in betamethasone raw materials by selecting a proper chromatographic column, adjusting the proportion of a mobile phase, the temperature and the flow rate of the column, setting gradient elution and utilizing a high-performance liquid chromatography.
(2) The method adopts a high-efficiency durable chromatographic method, impurities SM1r, SM1b, SM1d and SM1q which cannot be effectively separated by a betamethasone EP10.0 related substance analysis method can be well separated from main peaks of a sample and other impurities possibly existing in the sample within 37 minutes, and the problems of separation and measurement of the impurities SM1r, SM1b, SM1d and SM1q in the betamethasone which cannot be solved by the prior art are provided; the analysis method has high sensitivity; the specificity is strong; the repeatability is good; the operation is simple and feasible.
Drawings
FIG. 1 is an HPLC chart of a test solution of betamethasone measured by the EP10.0 method;
FIG. 2 is an HPLC chart of a test solution for determining betamethasone in the present invention;
FIG. 3 is an HPLC chart of a limiting solution of example 3 of the present invention;
FIG. 4 is an HPLC chart showing the chromatographic condition durability 2 of the sample mixed solution of example 4 of the present invention;
FIG. 5 is an HPLC chart showing the chromatographic condition durability 3 of the sample mixed solution of example 4 of the present invention;
FIG. 6 is an HPLC chart showing the chromatographic condition durability 4 of the sample mixed solution of example 4 of the present invention;
FIG. 7 is an HPLC chart showing the chromatographic condition durability 5 of the sample mixed solution of example 4 of the present invention;
FIG. 8 is an HPLC chart showing the chromatographic condition durability 6 of the sample mixed solution of example 4 of the present invention;
FIG. 9 is an HPLC chart showing the chromatographic condition durability 7 of the sample mixed solution of example 4 of the present invention;
FIG. 10 is an HPLC chart showing the chromatographic condition durability 8 of the sample mixed solution of example 4 of the present invention;
FIG. 11 is an HPLC chart showing the durability 9 of the chromatographic conditions of the sample mixed solution of example 4 of the present invention.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Example 1
Preparing a solution:
taking a proper amount of the product, precisely weighing, placing into a measuring flask, diluting with a diluent [ methanol-acetonitrile (50:50) ] to prepare a solution containing about 1.0mg in each 1ml, and shaking uniformly to obtain a sample solution.
The impurities SM1a, SM1b, SM1c, SM1d, SM1e, SM1g, SM1k, SM1l, SM1q, SM1r, 5-dimethylhydantoin were dissolved in a suitable amount of diluent and diluted to prepare a mixed solution containing about 1. Mu.g/ml per 1ml as a reference solution.
The method comprises the following steps:
high performance liquid chromatography using octadecylsilane chemically bonded silica as filler (Shim-Pack VP-ODS 4.6mm.times.250 mm,5 μm or column with equivalent performance); linear gradient elution is carried out by taking water as a mobile phase A, acetonitrile as a mobile phase B and methanol as a mobile phase C (see table 1), and the detection wavelength is 254nm; the flow rate is 1.0ml per minute; the column temperature was 30 ℃. Precisely measuring 10 μl of each of the sample solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. And calculating the impurity content in the sample solution according to an external standard method.
TABLE 1 gradient elution procedure
The betamethasone at the current stage has EP10.0, USP42S2 and JP17 pharmacopoeia methods, wherein the betamethasone EP10.0 related substance analysis method with better separation effect on known impurities and other related impurities has the advantages of separating impurities SM1r and SM 17 1b Cannot be effectively separated, and impurities SM1d and SM1q cannot reach the baseline separation (see fig. 1, table 2). By screening chromatographic columns and chromatographic conditions, an HPLC method is established, impurities SM1r and SM1b are effectively separated, and the separation effect of impurities SM1d and SM1q is better than that of betamethasone EP10.0 related substancesBetter (see figure 2, table 3).
TABLE 2 EP10.0 measurement of the integration results of betamethasone
TABLE 3 method of the invention for determining the integral results of the solutions of betamethasone test samples
Example 2
Specialization:
impurities that may be present in betamethasone: SM (SM) 1a 、SM 1c 、SM 1e 、SM 1g 、SM 1k 、SM 1l 、SM 1b 、SM 1d 、SM 1q 、SM 1r Total 11 impurities of 5, 5-dimethyl hydantoin, we refer to impurity SM 1b 、SM 1d 、SM 1q 、SM 1r Studies were performed to investigate the separation between these 4 impurities and the remaining 7 impurities.
Taking 10 μl of each of blank solution, impurity positioning solution, other impurity mixed solution, sample solution, and mixed solution, performing sample injection according to a law, recording a chromatogram (see figure 2), and determining the result in table 4.
TABLE 4 determination results of specificity test
Conclusion: blank solution does not interfere with impurity SM 1r 、SM 1b 、SM 1d And SM 1q Is detected; the test sample does not interfere with the detection of the impurities; the separation degree between other impurities possibly existing in the sample and the impurities and the separation degree between the impurities are all more than 1.5, and the method specificity meets the requirements.
Example 3
Limit of detection:
and taking the detection limit solution to continuously sample for 3 times, and calculating the ratio (signal to noise ratio) of the peak height of the main peak to the noise. The test results are shown in FIG. 3, tables 5 and 6.
TABLE 5 detection limit solution integration results
TABLE 6 detection limit measurement results
Conclusion: impurity SM 1r The detection limit concentration is 0.032 mug/ml, the concentration in the sample is 0.0032%, and the signal to noise ratio average value is 4.1; impurity SM 1b The detection limit concentration is 0.033 mug/ml, the concentration in the sample is 0.0033 percent, and the signal to noise ratio average value is 7.3; impurity SM 1d The detection limit concentration is 0.032 mug/ml, the concentration in the sample is 0.0032%, and the signal to noise ratio average value is 6.0; impurity SM 1q The detection limit concentration was 0.030. Mu.g/ml, the concentration was 0.0030% as the concentration present in the sample, and the signal to noise ratio average was 5.7.
Example 4
Chromatographic condition durability:
and (3) taking mixed solutions of the impurity samples, respectively using normal mobile phases, and testing different mobile phase proportions, column temperatures, column flow rates and chromatographic columns of the same type and different batches for the predetermined test, respectively testing after the instrument system is stable, and recording the separation degree between peaks, wherein the test results are shown in tables 7-15, and figures 2 and 4-11.
TABLE 7 test sample mixed solution chromatographic condition durability 2 integral results
TABLE 8 test sample mixed solution chromatographic condition durability 3 integration results
TABLE 9 test sample mixed solution chromatographic condition durability 4 integration results
TABLE 10 test sample mixed solution chromatographic condition durability 5 integration results
TABLE 11 test sample Mixed solution chromatographic condition durability 6 integral results
TABLE 12 test sample mixed solution chromatographic condition durability 7 integration results
TABLE 13 test sample Mixed solution chromatographic condition durability 8 integration results
TABLE 14 test sample Mixed solution chromatographic condition durability 9 integration results
TABLE 15 chromatographic condition durability measurement results
Conclusion: when the chromatographic conditions slightly fluctuate (the flow speed is +/-0.1 ml/min, the column temperature is +/-2 ℃, the acetonitrile initial proportion is +/-2%, the methanol initial proportion is +/-1%, and the chromatographic columns of different batches of the same type are basically consistent in the normalized content of the peak areas of all impurities, and the impurities SM 1r 、SM 1b 、SM 1d 、SM 1q The separation degree between the sample and the main peak of the sample and other impurities possibly existing in the sample is more than 1.5, and the durability of chromatographic conditions meets the requirements.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (7)
1. The method for separating the relevant impurities in the betamethasone raw material is characterized in that a chromatographic column is adopted, octadecylsilane chemically bonded silica is adopted as a filler, and a mobile phase A, a mobile phase B and a mobile phase C are adopted for gradient elution to separate the relevant impurities; the mobile phase A is water, the mobile phase B is acetonitrile, and the mobile phase C is methanol; the related impurities are an impurity composition consisting of SM1c, SM1e, SM1g and SM1k and any one or more of the following impurities: SM1a, SM1b, SM1d, SM1l, SM1q, SM1r, 5-dimethylhydantoin, the specific structural formula is as follows:
the specification of the chromatographic column is 4.6mm multiplied by 250mm and 5 mu m; the model of the chromatographic column is Shim-Pack VP-ODS; the gradient elution procedure was:
2. the method of claim 1, wherein the column temperature is 28-32 ℃.
3. The method according to claim 1, wherein the sample is introduced in an amount of 10 to 30. Mu.l.
4. The method according to claim 1, wherein the mobile phase flow rate is 0.9 to 1.1ml/min.
5. The method for detecting the relevant impurities in the betamethasone raw material is characterized in that the relevant impurities in the betamethasone raw material are separated by the method in claim 1, the betamethasone raw material is detected by a detector, the wavelength of the detector is set to 245-265 nm, a chromatogram of a sample to be detected is compared with a chromatogram of a known reference substance, and whether the betamethasone raw material contains the relevant impurities is judged, wherein the relevant impurities are the relevant impurities in claim 1.
6. A method for determining relevant impurities in a betamethasone feedstock, characterized in that the method comprises the specific steps of:
1) Preparing a test sample solution and a reference substance solution: dissolving a sample to be tested in a diluent to obtain a sample solution; dissolving the related impurity reference substance in a diluent to obtain a reference substance solution;
2) Separating: separating the impurities associated with the betamethasone feedstock by the method of claim 1;
3) And (3) detection: detecting the presence or absence of said related impurities in a starting material of dexamethasone using the method as described in claim 5;
4) And (3) measuring: calculating the content of the related impurities in the sample solution according to the detected chromatogram and the peak area by an external standard method; the related impurity is the related impurity described in claim 1.
7. The method of claim 6, wherein the diluent is methanol and acetonitrile in a volume ratio of 1:1.
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