CN119335077A - A method for analyzing related substances in estradiol dydrogesterone tablets - Google Patents
A method for analyzing related substances in estradiol dydrogesterone tablets Download PDFInfo
- Publication number
- CN119335077A CN119335077A CN202411351556.5A CN202411351556A CN119335077A CN 119335077 A CN119335077 A CN 119335077A CN 202411351556 A CN202411351556 A CN 202411351556A CN 119335077 A CN119335077 A CN 119335077A
- Authority
- CN
- China
- Prior art keywords
- dydrogesterone
- estradiol
- mobile phase
- impurity
- chromatographic column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Steroid Compounds (AREA)
Abstract
本发明公开了一种雌二醇地屈孕酮片有关物质的分析方法。本发明提供了一种雌二醇和/或地屈孕酮的检测分离方法，其包括如下步骤：采用液相色谱法对待测样品进行检测或分离；其中，所述液相色谱法的检测条件包括：色谱柱为十八烷基键合硅胶反相色谱柱；流动相A为水；流动相B为乙腈‑甲醇；流动相C为乙醇；所述流动相B中，所述乙腈和所述甲醇的体积比为1:1；所述液相色谱法的洗脱方式为线性梯度洗脱。本发明提供的检测分离方法可以使雌二醇地屈孕酮片工艺及降解杂质有效分离，并且操作简单，灵敏度高，专属性强，准确度好。The present invention discloses an analysis method of related substances of estradiol dydrogesterone tablets. The present invention provides a detection and separation method of estradiol and/or dydrogesterone, which comprises the following steps: using liquid chromatography to detect or separate the sample to be tested; wherein, the detection conditions of the liquid chromatography include: the chromatographic column is an octadecyl bonded silica gel reverse phase chromatographic column; mobile phase A is water; mobile phase B is acetonitrile-methanol; mobile phase C is ethanol; in the mobile phase B, the volume ratio of the acetonitrile and the methanol is 1:1; the elution mode of the liquid chromatography is linear gradient elution. The detection and separation method provided by the present invention can effectively separate the estradiol dydrogesterone tablet process and degradation impurities, and is simple to operate, highly sensitive, highly specific and accurate.
Description
Technical Field
The invention relates to a method for analyzing substances related to a estradiol dydrogesterone tablet.
Background
Estradiol (Estradiol), chemical name estra-1, 3,5 (10) -triene-3, 17 beta-diol, chemical structure:
Dydrogesterone (Dydrogesterone), chemical name 9 beta, 10 alpha-pregnane-4, 6-diene-3, 20-dione, chemical structure:
Estradiol is an artificially synthesized estrogen, and can replace estrogen reduction of climacteric women, and relieve climacteric symptoms.
Dydrogesterone is a progestogen with oral activity. As estrogen promotes endometrial growth, elevated estrogen levels will increase the risk of endometrial hyperplasia and cancer. The addition of a progestogen greatly reduces the risk of estrogen-induced endometrial hyperplasia in women without hysterectomy. The estradiol tablet/estradiol dydrogesterone tablet composite package adopts the combination of estrogens, improves the medication safety of perimenopausal syndrome treatment, and has positive effects on improving clinical curative effects.
Because the estradiol tablet/estradiol dydrogesterone tablet composite package comprises two tablets, namely the estradiol tablet and the estradiol dydrogesterone tablet, the raw materials, intermediates, reaction byproducts and the like possibly remain in the process from the synthesis of raw materials to the preparation of the dydrogesterone tablet, and degradation products can also be generated in the storage process. Therefore, an effective analysis method of the relevant substances of the estradiol dydrogesterone tablet is established, the determination of the relevant substances of the estradiol dydrogesterone tablet is rapidly and accurately realized, and the method has important practical significance in the aspects of synthesis and quality control of the preparation process.
In the imported drug registration standard JX20130310, 4 related substances of estradiol are disclosed as shown in table 1 below, in the imported drug registration standard JX20130310, 2 related substances of dydrogesterone (6-dydrogesterone and 17α -dydrogesterone) are disclosed, and in the european pharmacopoeia 11.0, dydrogesterone impurity a is also disclosed, and detailed information of each impurity is shown in table 2.
TABLE 1 substances related to the estradiol disclosures
。
TABLE 2 dydrogesterone disclosed related substances
。
However, after the imported registration criteria are reproduced, we find that the separation degree of 17 alpha-estradiol and estradiol is poor, the dydrogesterone impurity A is completely overlapped with dydrogesterone, the 6-dydrogesterone is completely overlapped with 17 alpha-dydrogesterone, and the situation that main peaks are not well separated from adjacent impurities and the dydrogesterone specific impurities are completely overlapped exists for the estradiol and the dydrogesterone, so that the detection conditions need to be explored and improved, and a set of analysis methods suitable for the analysis of the materials of the estradiol and the dydrogesterone and related single and compound preparations are established at the same time, and the analysis methods are used for quality control of raw medicines, intermediate products and finished products.
Disclosure of Invention
The invention solves the technical problem of providing a method for analyzing relevant substances of an estradiol dydrogesterone tablet by utilizing high performance liquid chromatography, which can effectively separate the process and degradation impurities (shown in tables 1 and 2 in detail) of the estradiol dydrogesterone tablet, and has the advantages of simple operation, effective detection of a plurality of impurities of the estradiol dydrogesterone tablet and a plurality of process impurities (loaded in EP) in raw medicines, high sensitivity and strong specificity.
The present invention solves the above technical problems by the following solution.
The invention provides a detection and separation method of estradiol and/or dydrogesterone, which comprises the following steps:
Detecting or separating a sample to be detected by adopting a liquid chromatography method, wherein the sample to be detected comprises a substance 1 and/or a substance 2, the substance 1 is estradiol and an impurity 1, the impurity 1 is one or more of 17 alpha-estradiol, estrone, delta 9, 11-estradiol and 6-keto-estradiol, the substance 2 is dydrogesterone and an impurity 2, and the impurity 2 is one or more of dydrogesterone impurities A, 6-dydrogesterone and 17 alpha-dydrogesterone;
wherein the detection conditions of the liquid chromatography include:
the chromatographic column is octadecyl bonded silica gel reversed phase chromatographic column;
mobile phase A is water;
Mobile phase B is acetonitrile-methanol;
Mobile phase C is ethanol;
in the mobile phase B, the volume ratio of the acetonitrile to the methanol is 1:1;
The elution mode of the liquid chromatography is linear gradient elution, and the elution program is as follows:
;
Wherein X1 is 62-64%, X2 is 50-52%, Y1 is 36-38%, Y2 is 10%, Y3 is 35%, in the table, "0.fwdarw.15 min, the volume of mobile phase A is X1", which means that mobile phase A (volume of mobile phase A is a percentage of the total volume of mobile phase) is fixed to X1 in the range of 0-15 min, "15.fwdarw.20 min, the volume of mobile phase B is Y1. Fwdarw.Y2", which means that mobile phase B (volume of mobile phase B is a percentage of the total volume of mobile phase) is linearly changed from Y1 to Y2 in the range of 15-20 min.
In some embodiments of the invention, the impurity 1 is selected from one or more of 17α -estradiol, estrone, and 6-keto-estradiol.
In some embodiments of the invention, the column type is ChromCore 120C 18 or thermo Hypersil ODS, preferably the column is ChromCore 120 C18 4.6*150mm,5 μm or thermo Hypersil ODS, 150X 4.6mm,3 μm.
In some embodiments of the invention, the packing particle size of the chromatographic column may be 2.5-7.5 μm, for example 5 μm or 3 μm.
In some embodiments of the invention, the chromatographic column may have a length of 100-200mm, preferably 150mm.
In some embodiments of the invention, the inner diameter of the chromatographic column may be 3-5mm, preferably 4.6mm.
In some embodiments of the invention, the elution procedure for the linear gradient elution is:
Or alternatively, the first and second heat exchangers may be,
。
In some embodiments of the present invention, the sample to be tested is pretreated to meet the sample injection standard before the test, where the pretreatment is conventional in the art, and specifically includes dissolving the sample to be tested in a diluent, where the diluent may be a mixed solution of acetonitrile and water, the volume ratio of acetonitrile to water is preferably 40:60, and the mass-volume ratio of each substance in the sample to the diluent may be 0.01-500 μg/mL, preferably 0.1-500 μg/mL, for example 0.13 μg/mL, 0.15 μg/mL, 0.3 μg/mL, 0.5 μg/mL, 100 μg/mL or 500 μg/mL. Preferably, after the dissolving step, a filtration step is further included, for example filtration using a polytetrafluoroethylene microporous membrane.
In some embodiments of the invention, the column temperature of the chromatographic column may be 35-45 ℃, preferably 40 ℃.
In some embodiments of the present invention, the detector of the detection separation method may be an ultraviolet detector, the detection wavelength of the detector may be 220nm when the sample to be detected contains substance 1, and the detection wavelength of the detector may be 310nm when the sample to be detected contains substance 2.
In some embodiments of the invention, the flow rate of the mobile phase may be in reference to conventional flow rates as determined by chromatography in the art, for example 0.8-1.2 mL/min, preferably 1.0 mL/min.
In some embodiments of the invention, the liquid chromatography sample may be performed with reference to conventional sample amounts for detection analysis by chromatography in the art, for example 20-80. Mu.L, preferably 50. Mu.L.
In some embodiments of the invention, the test sample comprises dydrogesterone and dydrogesterone impurity a.
In some embodiments of the invention, the sample to be tested comprises estradiol and 17α -estradiol.
In some embodiments of the invention, the test sample may further comprise 6-dydrogesterone and 17 a-dydrogesterone.
In some embodiments of the invention, the test sample consists of 17α -estradiol, estrone, estradiol, 6-keto-estradiol, dydrogesterone impurity a, 6-dydrogesterone, and 17α -dydrogesterone.
The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
the method is suitable for separating and measuring the impurities of the estradiol, the dydrogesterone and the related single and compound preparations, can be used for independently detecting a certain substance, and can also be used for simultaneously separating and detecting the estradiol, the dydrogesterone and the related impurities. Compared with the prior art, the detection separation method has better separation effect on estradiol and related impurities, dydrogesterone and related impurities, and has the advantages of strong specificity, higher sensitivity and good accuracy, and the tablet auxiliary materials do not interfere with detection, so that the quality of the estradiol dydrogesterone tablet can be better controlled, and the method has obvious progress.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The experimental apparatus used in the following examples was either Shimadzu or Siemens high performance liquid chromatograph (UV detector), the mobile phase A was water, the mobile phase B was acetonitrile-methanol (1:1), the mobile phase C was ethanol, gradient elution was performed, the column temperature was 40 ℃, the flow rate was 1.0ml/min, the solvent used for diluting the sample was acetonitrile-water (40:60), the sample injection volume was 50. Mu.l, the detection wavelength was 220nm (estradiol and related impurities), 310nm (dydrogesterone and related impurities).
The sample and control sources involved in the examples are as follows (table 3):
TABLE 3 information table of samples and controls
。
EXAMPLE 1 determination of the substances associated with the oestradiol dydrogesterone tablet
Chromatographic conditions:
ChromCore 120C 18 μm, 4.6X105 mm
Mobile phase A is water, mobile phase B is acetonitrile-methanol (1:1), mobile phase C is ethanol;
Gradient elution is as follows:
Wherein the elution gradient varies linearly, e.g., 42-50 minutes in the table above, mobile phase a (volume of mobile phase a as a percentage of the total volume of mobile phase) varies linearly from 62% to 50%, while mobile phase B (volume of mobile phase B as a percentage of the total volume of mobile phase) varies linearly from 10% to 35%, mobile phase C (volume of mobile phase C as a percentage of the total volume of mobile phase) varies linearly from 28% to 15%, wherein the sum of mobile phase a, mobile phase B and mobile phase C is always 100%. The remaining embodiments are not explained here.
The impurity mixed solution is prepared by respectively weighing 6-ketone-estradiol, 17 alpha-estradiol, estrone and dydrogesterone impurity A, 6-dydrogesterone, 17 alpha-dydrogesterone and dydrogesterone tablet powder with proper amounts, diluting with solvent (acetonitrile-water (40:60)) to prepare a solution containing about 0.5 mug of each impurity, 100 mug of estradiol and 500 mug of dydrogesterone in each 1ml, mixing uniformly and filtering.
The retention time, the degree of separation, etc. were recorded by precisely measuring 50. Mu.l of the mixed impurity solution injected into the liquid chromatograph. The results of each peak, as compared to the standard, are shown in table 4:
TABLE 4 Table 4
。
As shown by the results of the table, the analysis method can effectively detect each impurity of estradiol under the detection condition of 220nm, and can effectively detect each impurity of dydrogesterone under the detection condition of 310 nm.
EXAMPLE 2 determination of the substances associated with the oestradiol dydrogesterone tablet
Chromatographic conditions:
ChromCore 120C 18 μm, 4.6X105 mm
Mobile phase A is water, mobile phase B is acetonitrile-methanol (1:1), mobile phase C is ethanol;
Gradient elution is as follows:
。
The impurity mixed solution is prepared by respectively weighing 6-ketone-estradiol, 17 alpha-estradiol, estrone and dydrogesterone impurity A, 6-dydrogesterone, 17 alpha-dydrogesterone and dydrogesterone tablet powder with proper amounts, diluting with solvent (acetonitrile-water (40:60)) to prepare a solution containing about 0.5 mug of each impurity, 100 mug of estradiol and 500 mug of dydrogesterone in each 1ml, mixing uniformly and filtering.
The retention time, the degree of separation, etc. were recorded by precisely measuring 50. Mu.l of the mixed impurity solution injected into the liquid chromatograph. The results of each peak are shown in table 5, against the standard control:
TABLE 5
。
As shown by the results of the table, the analysis method can effectively detect each impurity of estradiol under the detection condition of 220nm, and can effectively detect each impurity of dydrogesterone under the detection condition of 310 nm.
EXAMPLE 3 determination of the substances associated with the oestradiol dydrogesterone tablet
Chromatographic conditions:
Thermo Hypersil ODS, 150X 4.6mm,3 μm
Mobile phase A is water, mobile phase B is acetonitrile-methanol (1:1), mobile phase C is ethanol;
Gradient elution is as follows:
。
The impurity mixed solution is prepared by respectively weighing 6-ketone-estradiol, 17 alpha-estradiol, estrone and dydrogesterone impurity A, 6-dydrogesterone, 17 alpha-dydrogesterone and dydrogesterone tablet powder with proper amounts, diluting with solvent (acetonitrile-water (40:60)) to prepare a solution containing about 0.5 mug 6-ketone-estradiol, 0.3 mug 17 alpha-estradiol, 0.5 mug estrone, 0.5 mug dydrogesterone impurity A, 0.13 mug 6-dydrogesterone, 0.15 mug 17 alpha-dydrogesterone, 100 mug estradiol and 500 mug dydrogesterone in each 1ml, mixing and filtering to obtain the impurity mixed solution.
The retention time, the degree of separation, etc. were recorded by precisely measuring 50. Mu.l of the mixed impurity solution injected into the liquid chromatograph. The results of each peak are shown in table 6, against a standard control:
TABLE 6
。
As shown by the results of the table, the analysis method can effectively detect each impurity of estradiol under the detection condition of 220nm, and can effectively detect each impurity of dydrogesterone under the detection condition of 310 nm.
EXAMPLE 4 Pre-experiment of the accuracy of the method for producing the estramustine tablet related substances
Respectively taking 5 estradiol tablets (22240506) and placing the estradiol tablets into a 100ml measuring flask, adding a proper amount of solvent (acetonitrile-water (40:60)), carrying out ultrasonic treatment for 15 minutes to dissolve the estradiol tablets, cooling, respectively adding a proper amount of impurity mixed solution into the measuring flask, diluting to a scale with the solvent, shaking uniformly, centrifuging at 2500rpm of a centrifuge for 10 minutes, filtering with a 0.45 mu m polytetrafluoroethylene filter membrane, and taking filtrate as a series of sample solutions with accuracy. Under the established chromatographic conditions (same as in example 1), sample injection analysis is carried out on the sample solution, the detection amount of each substance is calculated by an external standard method (estradiol is used as an external standard for the estradiol related impurities, dydrogesterone is used as an external standard for the dydrogesterone related impurities), and the recovery rate is calculated by the ratio of the actual measurement result (detection amount) to the theoretical value, and the result is shown in Table 7.
TABLE 7
。
Relative correction factor for estradiol related impurities:
TABLE 8
Relative correction factors for dydrogesterone related impurities:
TABLE 9
。
Conclusion that the recovery rate of each impurity of the estradiol and the dydrogesterone is qualified (the acceptable standard is 90.0% -108.0%) within the concentration range of 20% -150%.
EXAMPLE 5 method of detecting limit and quantitative item of substances related to the oestradiol dydrogesterone tablet
And (3) respectively dissolving estradiol, dydrogesterone and related impurities by using a solvent, gradually diluting, respectively carrying out sample injection analysis on the diluted solution under the planned chromatographic conditions (same as in example 1), recording a chromatogram, establishing a quantitative limit according to a signal-to-noise ratio S/N (10/1), and establishing a detection limit according to a signal-to-noise ratio S/N (3/1). The results are shown in Table 10:
Table 10 limit of detection and limit of quantification test results
Note that the test sample concentration was 0.1mg/ml for estradiol and related impurities and 0.5mg/ml for dydrogesterone and related impurities.
The conclusion is that the detection limit and the quantitative limit of each component are lower than 0.05% of the concentration of the sample, each impurity can be effectively detected and accurately quantified, and the sensitivity of the method is good.
Comparative example 1 determination of the substances related to the tablet of estramustine
The test method comprises the steps of measuring according to an import registration standard JX20130310 method, wherein the mobile phase is acetonitrile-water (425:600), isocratic elution, the flow rate is 1.0ml/min, a chromatographic column is thermo Hypersil ODS,150 multiplied by 4.6mm and 3 mu m, the column temperature is 40 ℃, the sample injection volume is 20 mu l, the running time is 40 minutes, and the detection wavelength is 220nm (estradiol and related impurities) and 310nm (dydrogesterone and related impurities). The solvent used to dilute the sample was acetonitrile-water (40:60).
And (3) respectively weighing 6-ketone-estradiol, 17 alpha-estradiol, estrone and dydrogesterone impurities A, 6-dydrogesterone, 17 alpha-dydrogesterone and estradiol dydrogesterone tablet powder in proper amounts, diluting with a solvent to prepare a solution containing about 0.5 mug of each impurity, 100 mug of estradiol and 500 mug of dydrogesterone in 1ml, uniformly mixing and filtering to obtain the impurity mixed solution.
20 Μl of the impurity mixed solution was precisely measured and injected into a liquid chromatograph to record retention time, separation degree, etc. The results of each peak are shown in table 11, against the standard control:
TABLE 11
。
The results show that the detection of 6-keto-estradiol is interfered, the separation of estradiol and adjacent impurities is poor, and the effective separation between dydrogesterone and dydrogesterone impurity A and between 6-dydrogesterone and 17 alpha-dydrogesterone cannot be realized.
Comparative example 2 determination of the substances related to the estramustine tablet
The test method comprises the steps of referring to a method of European pharmacopoeia estradiol related substances, taking acetonitrile-ethanol (96) -water (21:25:54) as a mobile phase, carrying out isocratic elution, carrying out flow rate of 1.0ml/min, carrying out chromatographic column, namely Phenomnex Luna C18 (2) 100A,4.6mm 25cm and 5 mu m, carrying out column temperature of 40 ℃, carrying out sample injection volume of 20 mu l, carrying out operation time of 60 minutes, and carrying out detection wavelength of 280nm. The solvent used to dilute the sample was acetonitrile-water (40:60).
And (3) respectively weighing 6-ketone-estradiol, 17 alpha-estradiol, estrone and dydrogesterone impurities A, 6-dydrogesterone, 17 alpha-dydrogesterone and estradiol dydrogesterone tablet powder in proper amounts, diluting with a solvent to prepare a solution containing about 0.5 mug of each impurity, 100 mug of estradiol and 500 mug of dydrogesterone in 1ml, uniformly mixing and filtering to obtain the impurity mixed solution.
20 Μl of the impurity mixed solution was precisely measured and injected into a liquid chromatograph to record retention time, separation degree, etc. The results of each peak, as compared to the standard, are shown in table 12:
Table 12
。
The results show that the 6-keto-estradiol response value is low and that an effective separation between dydrogesterone and dydrogesterone impurity a, 6-dydrogesterone and 17 alpha-dydrogesterone cannot be achieved in this method.
Comparative example 3 determination of the substances related to the estramustine tablet
Test methods referring to the United states pharmacopoeia dydrogesterone related substances method, the mobile phase was acetonitrile-methanol-water (400:50:550), isocratic elution, flow rate of 1.0ml/min, chromatographic column: thermo Hypersil ODS, 150X 4.6mm,3 μm, column temperature of 40 ℃, sample injection volume of 20. Mu.l, run time of 60 min, detection wavelength of 280nm.
The impurity mixed solution is prepared by respectively weighing 6-ketone-estradiol, 17 alpha-estradiol, estrone and dydrogesterone impurity A, 6-dydrogesterone, 17 alpha-dydrogesterone and dydrogesterone tablet powder with proper amounts, diluting with solvent to prepare a solution containing about 0.5 mug of 6-ketone-estradiol, 0.3 mug of 17 alpha-estradiol, 0.5 mug of estrone, 0.5 mug of dydrogesterone impurity A, 0.13 mug of 6-dydrogesterone, 0.15 mug of 17 alpha-dydrogesterone, 100 mug of estradiol and 500 mug of dydrogesterone in each 1ml, mixing evenly and filtering to obtain the impurity mixed solution.
20 Μl of the impurity mixed solution was precisely measured and injected into a liquid chromatograph to record retention time, separation degree, etc. The results of each peak are shown in table 13, against the standard:
TABLE 13
。
The results show that the method has low response values of various impurities of estradiol, the separation of estrone and 17 alpha-estradiol is poor, and the effective separation of dydrogesterone and dydrogesterone impurity A cannot be realized.
Comparative example 4 determination of the substances related to the oestradiol dydrogesterone tablet
Chromatographic conditions:
Thermo Hypersil ODS, 150X 4.6mm,3 μm
Mobile phase A is water, mobile phase B is acetonitrile, mobile phase C is ethanol;
Gradient elution is as follows:
The impurity mixed solution is prepared by respectively weighing 6-ketone-estradiol, 17 alpha-estradiol, estrone and dydrogesterone impurity A, 6-dydrogesterone, 17 alpha-dydrogesterone and dydrogesterone tablet powder with proper amounts, diluting with solvent to prepare a solution containing about 0.5 mug of 6-ketone-estradiol, 0.3 mug of 17 alpha-estradiol, 0.5 mug of estrone, 0.5 mug of dydrogesterone impurity A, 0.13 mug of 6-dydrogesterone, 0.15 mug of 17 alpha-dydrogesterone, 100 mug of estradiol and 500 mug of dydrogesterone in each 1ml, mixing evenly and filtering to obtain the impurity mixed solution.
20 Μl of the impurity mixed solution was precisely measured and injected into a liquid chromatograph to record retention time, separation degree, etc. The results of each peak, as compared to the standard, are shown in table 14:
TABLE 14
。
As shown by the results of the table, the analysis method can effectively detect each impurity of the estradiol under the detection condition of 220nm, but the baseline fluctuation is large, the integral of the estrone is easy to be interfered, and the dydrogesterone impurity A and the dydrogesterone peak are completely overlapped and cannot be separated under the detection condition of 310 nm.
Comparative example 5 determination of the substances related to the oestradiol dydrogesterone tablet
Chromatographic conditions:
Thermo Hypersil ODS, 150X 4.6mm,3 μm
Mobile phase A is water, mobile phase B is acetonitrile-methanol (4:1), mobile phase C is ethanol;
Gradient elution is as follows:
The impurity mixed solution is prepared by respectively weighing 6-ketone-estradiol, 17 alpha-estradiol, estrone and dydrogesterone impurity A, 6-dydrogesterone, 17 alpha-dydrogesterone and dydrogesterone tablet powder with proper amounts, diluting with solvent to prepare a solution containing about 0.5 mug of 6-ketone-estradiol, 0.3 mug of 17 alpha-estradiol, 0.5 mug of estrone, 0.5 mug of dydrogesterone impurity A, 0.13 mug of 6-dydrogesterone, 0.15 mug of 17 alpha-dydrogesterone, 100 mug of estradiol and 500 mug of dydrogesterone in each 1ml, mixing evenly and filtering to obtain the impurity mixed solution.
20 Μl of the impurity mixed solution was precisely measured and injected into a liquid chromatograph to record retention time, separation degree, etc. The results of each peak, as compared to the standard, are shown in table 15:
TABLE 15
。
As shown by the results of the table, the analysis method can effectively detect each impurity of the estradiol under the detection condition of 220nm, but the baseline fluctuation is large, the integral of 17 alpha-estradiol and estrone is easy to be interfered, and the dydrogesterone impurity A and the dydrogesterone peak are basically completely overlapped and cannot be separated under the detection condition of 310 nm.
Claims (10)
1. A method for detecting and separating estradiol and/or dydrogesterone, which is characterized by comprising the following steps:
Detecting or separating a sample to be detected by adopting a liquid chromatography method, wherein the sample to be detected comprises a substance 1 and/or a substance 2, the substance 1 is estradiol and an impurity 1, the impurity 1 is one or more of 17 alpha-estradiol, estrone, delta 9, 11-estradiol and 6-keto-estradiol, the substance 2 is dydrogesterone and an impurity 2, and the impurity 2 is one or more of dydrogesterone impurities A, 6-dydrogesterone and 17 alpha-dydrogesterone;
wherein the detection conditions of the liquid chromatography include:
the chromatographic column is octadecyl bonded silica gel reversed phase chromatographic column;
mobile phase A is water;
Mobile phase B is acetonitrile-methanol;
Mobile phase C is ethanol;
in the mobile phase B, the volume ratio of the acetonitrile to the methanol is 1:1;
The elution mode of the liquid chromatography is linear gradient elution, and the elution program is as follows:
;
Wherein, X1 is 62-64%, X2 is 50-52%, Y1 is 36-38%, Y2 is 10%, Y3 is 35%.
2. The assay separation of claim 1, wherein the elution procedure for the linear gradient elution is:
Or alternatively, the first and second heat exchangers may be,
。
3. The assay separation method of claim 1, which satisfies one or more of the following conditions:
(1) The impurity 1 is selected from one or more of 17 alpha-estradiol, estrone and 6-keto-estradiol;
(2) The chromatographic column is ChromCore 120,120C 18 or thermo Hypersil ODS;
(3) The filler particle size of the chromatographic column is 2.5-7.5 mu m;
(4) The length of the chromatographic column is 100-200mm;
(5) The inner diameter of the chromatographic column is 3-5mm;
(6) The column temperature of the chromatographic column is 35-45 ℃;
(7) The flow rate of the mobile phase is 0.8-1.2 mL/min;
(8) The sample injection amount of the liquid chromatography is 20-80 mu L.
4. A detection separation method as claimed in claim 3, which satisfies one or more of the following conditions:
(1) The packing particle size of the chromatographic column is 5 μm or 3 μm;
(2) The length of the chromatographic column is 150mm;
(3) The inner diameter of the chromatographic column is 4.6mm;
(4) The column temperature of the chromatographic column is 40 ℃;
(5) The flow rate of the mobile phase is 1.0 mL/min;
(6) The sample injection amount of the liquid chromatography was 50. Mu.L.
5. A detection separation method according to claim 3, wherein the chromatographic column is ChromCore 120 C18 4.6*150mm,5 μm or thermo Hypersil ODS,150 x 4.6mm,3 μm.
6. The detection separation method according to claim 1, wherein the detector of the detection separation method is an ultraviolet detector;
Preferably, when the sample to be detected contains substance 1, the detection wavelength of the detector is 220nm;
and/or, when the substance 2 is contained in the sample to be detected, the detection wavelength of the detector is 310nm.
7. The method for detecting and separating according to claim 1, comprising the steps of pretreating the sample to be detected before the detection;
The pretreatment comprises the steps of dissolving the sample to be tested in a diluent, wherein the diluent is a mixed solution of acetonitrile and water, and preferably, the volume ratio of the acetonitrile to the water is 40:60.
8. The method according to claim 7, wherein the mass/volume ratio of each substance in the sample to be tested to the diluent is 0.01-500. Mu.g/mL, preferably 0.1-500. Mu.g/mL, such as 0.13. Mu.g/mL, 0.15. Mu.g/mL, 0.3. Mu.g/mL, 0.5. Mu.g/mL, 100. Mu.g/mL or 500. Mu.g/mL.
9. The method of claim 8, further comprising a step of filtering after the dissolving step, for example, by using a polytetrafluoroethylene microporous membrane.
10. The assay separation method of claim 1, wherein the sample to be tested consists of 17α -estradiol, estrone, estradiol, 6-keto-estradiol, dydrogesterone impurity a, 6-dydrogesterone, and 17α -dydrogesterone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411351556.5A CN119335077A (en) | 2024-09-26 | 2024-09-26 | A method for analyzing related substances in estradiol dydrogesterone tablets |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411351556.5A CN119335077A (en) | 2024-09-26 | 2024-09-26 | A method for analyzing related substances in estradiol dydrogesterone tablets |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN119335077A true CN119335077A (en) | 2025-01-21 |
Family
ID=94269301
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411351556.5A Pending CN119335077A (en) | 2024-09-26 | 2024-09-26 | A method for analyzing related substances in estradiol dydrogesterone tablets |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119335077A (en) |
-
2024
- 2024-09-26 CN CN202411351556.5A patent/CN119335077A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113049699B (en) | Method for detecting biphenyl anhydride and related substances thereof and application | |
| CN109633063B (en) | A kind of detection method of ticagrelor and its active metabolite concentration in human plasma | |
| CN113514569A (en) | Method for simultaneously measuring multiple endogenous hormones and exogenous hormones | |
| CN115248260A (en) | HPLC analysis detection method for related substances in cisatracurium besilate bulk drug | |
| CN108181386B (en) | Method for separating and measuring related substances in abiraterone acetate intermediate | |
| CN119335077A (en) | A method for analyzing related substances in estradiol dydrogesterone tablets | |
| CN105987961A (en) | Detection method of levetiracetam in breast milk | |
| CN110456087B (en) | Sertraline detection reagent and preparation and use methods thereof | |
| CN115236255B (en) | Method for detecting related substances of loxoprofen sodium | |
| CN106290600B (en) | A method of with liquid chromatography, separation health Buddhist nun replaces Buddhist nun and related substance | |
| CN110095554B (en) | Method for analyzing milrinone related substances by high performance liquid chromatography | |
| CN115436532A (en) | Detection method of sitaxel intermediate 5, 7-dichloro-1, 2,3, 4-tetrahydroisoquinoline | |
| CN110412164B (en) | Method for detecting related substances of mexiletine hydrochloride | |
| CN114354801A (en) | Method for analyzing content of three aminopyridine isomers in (R) -3-Boc-aminopiperidine | |
| CN114942278B (en) | Analysis method of related substances of Leitemivir intermediate di-D- (+) -di-p-methylbenzoyl tartaric acid ethyl acetate complex | |
| CN112250657B (en) | A kind of bicyclic alcohol dimer, its preparation method and use | |
| CN115856141B (en) | Method for separating and measuring tinib key intermediate and relevant impurities thereof by HPLC | |
| CN113092632B (en) | Method for detecting content of dehydroandrographolide in Chuanwang anti-inflammatory tablet | |
| CN116183758B (en) | Analysis method for isomer impurities in diethyl indenamine hydrochloride | |
| CN118501322B (en) | Method for detecting prednisolone sodium succinate related substances for injection | |
| CN117871705A (en) | Analysis method of estradiol valerate tablet related substances | |
| CN117630195A (en) | High performance liquid chromatography analysis method for calcitol related substances | |
| CN116678982B (en) | Detection method of paliperidone palmitate impurity SM1-G | |
| CN114965770B (en) | Method for detecting starting material, impurity D and impurity F in ifosfamide bulk drug | |
| CN114184712B (en) | Method for detecting content of free steroid impurities in conjugated estrogens tablets |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |