Analysis method of estradiol didrogesterone tablet related substances

Abstract

The invention discloses a method for analyzing related substances of estradiol didrogesterone tablets. The invention provides a method for detecting and separating estradiol and / or didrogesterone. The method comprises the following steps: detecting or separating a sample to be detected by adopting a liquid chromatography; wherein the detection conditions of the liquid chromatography are as follows: a chromatographic column is an octadecyl bonded silica gel reversed-phase chromatographic column; the mobile phase A is water; the mobile phase B is acetonitrile-methanol; a 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. According to the detection and separation method provided by the invention, an estradiol didrogesterone tablet process and degraded impurities can be effectively separated, and the method is simple to operate, high in sensitivity, strong in specificity and good in accuracy.

Description

Technical Field

[0001] The invention relates to an analysis method for related substances of estradiol dydrogesterone tablets. Background Art

[0002] Estradiol, chemical name: estradiol-1,3,5(10)-triene-3,17β-diol, chemical structure:

[0003]

[0004] Dydrogesterone, chemical name: 9β,10α-pregnane-4,6-diene-3,20-dione, chemical structure:

[0005]

[0006] Estradiol is a synthetic estrogen that can replace the decreased estrogen in menopausal women and alleviate menopausal symptoms.

[0007] Dydrogesterone is an orally active progestin. Because estrogen promotes the growth of the endometrium, elevated estrogen levels will increase the risk of endometrial hyperplasia and cancer. Adding progestin can greatly reduce the risk of estrogen-induced endometrial hyperplasia in women who have not had their uterus removed. The combined package of estradiol tablets / estradiol dydrogesterone tablets uses a combination of estrogen and progestin, which improves the safety of medication for the treatment of perimenopausal syndrome and has a positive effect on improving clinical efficacy.

[0008] Since the composite package of estradiol tablets / estradiol dydrogesterone tablets includes two tablets, estradiol tablets and estradiol dydrogesterone tablets, there may be residual starting materials, intermediates and reaction by-products in the process from the synthesis of raw materials to the preparation of estradiol and dydrogesterone, and degradation products may also be produced during the storage process. Therefore, it is of great practical significance to establish an effective analysis method for related substances of estradiol dydrogesterone tablets and quickly and accurately determine the related substances of estradiol dydrogesterone tablets in the quality control of the synthesis and preparation process.

[0009] Four related substances of estradiol are disclosed in the imported drug registration standard JX20130310, as shown in Table 1 below; two related substances of dydrogesterone (6-dehydroprogesterone and 17α-dydrogesterone) are disclosed in the imported drug registration standard JX20130310. In addition, dydrogesterone impurity A is also disclosed in the European Pharmacopoeia 11.0. The detailed information of each impurity is shown in Table 2.

[0010] Table 1: Disclosed related substances of estradiol

[0011] .

[0012] Table 2: Disclosed related substances of dydrogesterone

[0013] .

[0014] However, after reproducing the imported registration standards, we found that the separation degree between 17α-estradiol and estradiol was poor; the dydrogesterone impurity A completely overlapped with dydrogesterone, and 6-dehydroprogesterone completely overlapped with 17α-dydrogesterone. For estradiol and dydrogesterone, the main peak was poorly separated from the adjacent impurities and the specific impurities of dydrogesterone completely overlapped. Therefore, it was necessary to explore and improve the determination conditions; at the same time, a set of analytical methods suitable for estradiol, dydrogesterone raw materials and related single and compound preparations related substances should be established for the quality control of raw materials, intermediates and finished products. Summary of the invention

[0015] The technical problem solved by the present invention is to provide a method for analyzing related substances of estradiol dydrogesterone tablets by using high performance liquid chromatography. The method can effectively separate the process and degradation impurities of estradiol dydrogesterone tablets (see Tables 1 and 2 above for details), and the operation is simple. Multiple impurities of estradiol dydrogesterone tablets and several process impurities in raw materials (listed in EP) can be effectively detected with high sensitivity and strong specificity.

[0016] The present invention solves the above technical problems through the following solutions.

[0017] The present invention provides a method for detecting and separating estradiol and / or dydrogesterone, which comprises the following steps:

[0018] Liquid chromatography is used to detect or separate the sample to be tested; the sample to be tested includes substance 1 and / or substance 2, the substance 1 is estradiol and impurity 1, the impurity 1 is selected from one or more of 17α-estradiol, estrone, Δ9,11-estradiol and 6-keto-estradiol, the substance 2 is dydrogesterone and impurity 2, the impurity 2 is selected from one or more of dydrogesterone impurity A, 6-dehydroprogesterone and 17α-dydrogesterone;

[0019] Wherein, the detection conditions of the liquid chromatography method include:

[0020] The chromatographic column is an octadecyl bonded silica reverse phase chromatographic column;

[0021] Mobile phase A was water;

[0022] Mobile phase B was acetonitrile-methanol;

[0023] Mobile phase C was ethanol;

[0024] In the mobile phase B, the volume ratio of the acetonitrile to the methanol is 1:1;

[0025] The elution mode of the liquid chromatography is linear gradient elution, and the elution procedure is as follows:

[0026] ;

[0027] Among them, X1 is 62-64%; X2 is 50-52%; Y1 is 36-38%; Y2 is 10%; Y3 is 35%; in the table, for example, "0→15 minutes, the volume of mobile phase A is X1" means: in the range of 0-15 minutes, mobile phase A (the volume of mobile phase A is the percentage of the total volume of mobile phase) is fixed at X1; "15→20 minutes, the volume of mobile phase B is Y1→Y2" means that in the range of 15-20 minutes, mobile phase B (the volume of mobile phase B is the percentage of the total volume of mobile phase) changes linearly from Y1 to Y2.

[0028] In some embodiments of the present invention, the impurity 1 is selected from one or more of 17α-estradiol, estrone and 6-keto-estradiol.

[0029] In some embodiments of the present invention, the type of the chromatographic column is ChromCore 120 C18 or thermoHypersil ODS; preferably, the chromatographic column is ChromCore 120 C18 4.6*150 mm, 5 μm or thermoHypersil ODS, 150×4.6 mm, 3 μm.

[0030] In some embodiments of the present invention, the filler particle size of the chromatography column may be 2.5-7.5 μm, such as 5 μm or 3 μm.

[0031] In some embodiments of the present invention, the length of the chromatography column may be 100-200 mm, preferably 150 mm.

[0032] In some embodiments of the present invention, the inner diameter of the chromatography column may be 3-5 mm, preferably 4.6 mm.

[0033] In some embodiments of the present invention, the elution program of the linear gradient elution is:

[0034]

[0035] or,

[0036] .

[0037] In some embodiments of the present invention, before the test, the sample to be tested is pretreated to meet the injection standard, and the pretreatment is a conventional pretreatment in the art, specifically comprising: dissolving the sample to be tested in a diluent; the diluent may be a mixed solution of acetonitrile and water; the volume ratio of the acetonitrile and water is preferably 40:60; the mass volume ratio of each substance in the sample to be tested to the diluent may be 0.01~500 μg / mL; preferably 0.1~500μg / mL; for example 0.13μg / mL, 0.15μg / mL, 0.3μg / mL, 0.5μg / mL, 100μg / mL or 500μg / mL. Preferably, after the dissolution step, a filtration step is further included, for example, filtration is performed using a polytetrafluoroethylene microporous filter membrane.

[0038] In some embodiments of the present invention, the column temperature of the chromatographic column may be 35-45°C, preferably 40°C.

[0039] In some embodiments of the present invention, the detector of the detection and separation method may be an ultraviolet detector; when the sample to be tested contains substance 1, the detection wavelength of the detector may be 220nm; when the sample to be tested contains substance 2, the detection wavelength of the detector may be 310nm.

[0040] In some embodiments of the present invention, the flow rate of the mobile phase can refer to the conventional flow rate in chromatography detection in the art, such as 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0041] In some embodiments of the present invention, the injection volume of the liquid chromatography method can refer to the conventional injection volume of chromatography detection and analysis in the art, such as 20-80 μL, preferably 50 μL.

[0042] In some embodiments of the present invention, the test sample comprises dydrogesterone and dydrogesterone impurity A.

[0043] In some embodiments of the present invention, the test sample contains estradiol and 17α-estradiol.

[0044] In some embodiments of the present invention, the test sample may further contain 6-dehydroprogesterone and 17α-dydrogesterone.

[0045] In some embodiments of the present invention, the test sample consists of 17α-estradiol, estrone, estradiol, 6-keto-estradiol, dydrogesterone, dydrogesterone impurity A, 6-dehydroprogesterone and 17α-dydrogesterone.

[0046] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0047] The reagents and raw materials used in the present invention are commercially available.

[0048] The positive and progressive effects of the present invention are:

[0049] The method of the present invention is applicable to the separation and determination of estradiol, dydrogesterone and related single and compound preparation impurities, and can be used to detect a certain substance alone, or to simultaneously separate and detect estradiol, dydrogesterone and related impurities. Compared with the prior art, the detection and separation method of the present invention has a better separation effect on estradiol and related impurities, dydrogesterone and related impurities, has strong specificity, higher sensitivity, good accuracy, and the tablet excipients do not interfere with the detection, and can better control the quality of estradiol dydrogesterone tablets, which has significant progress. DETAILED DESCRIPTION

[0050] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0051] The experimental instruments used in the following examples are all Shimadzu or Thermo Fisher high performance liquid chromatographs (UV detector). Unless otherwise specified, mobile phase A is water, mobile phase B is acetonitrile-methanol (1:1), and mobile phase C is ethanol, and gradient elution is performed; the column temperature is 40°C; the flow rate is 1.0 ml / min; the solvent used for diluting the sample is acetonitrile-water (40:60); the injection volume is 50 μl; and the detection wavelengths are 220 nm (estradiol and related impurities) and 310 nm (dydrogesterone and related impurities).

[0052] The sources of samples and reference substances involved in the examples are as follows (Table 3):

[0053] Table 3: Sample and reference material information

[0054] .

[0055] Example 1 Determination of Related Substances in Estradiol Dydrogesterone Tablets

[0056] Chromatographic conditions:

[0057] Chromatographic column: ChromCore 120 C18 5μm, 4.6*150mm

[0058] Mobile phase: Mobile phase A is water; Mobile phase B is acetonitrile-methanol (1:1); Mobile phase C is ethanol;

[0059] Gradient elution:

[0060]

[0061] Wherein, the elution gradient changes linearly, for example, in the above table, from 42 to 50 minutes, the mobile phase A (the percentage of the volume of the mobile phase A to the total volume of the mobile phase) changes linearly from 62% to 50%, and at the same time, the mobile phase B (the percentage of the volume of the mobile phase B to the total volume of the mobile phase) changes linearly from 10% to 35%, and the mobile phase C (the percentage of the volume of the mobile phase C to the total volume of the mobile phase) changes linearly from 28% to 15%, wherein the sum of the mobile phase A, the mobile phase B and the mobile phase C is always 100%. The rest of the embodiments are the same and will not be explained again.

[0062] Impurity mixed solution: Weigh appropriate amounts of 6-keto-estradiol, 17α-estradiol, estrone and dydrogesterone impurity A, 6-dehydroprogesterone, 17α-dydrogesterone and estradiol dydrogesterone tablet powder respectively, dilute with solvent (acetonitrile-water (40:60)) to prepare a solution containing approximately 0.5 μg of each impurity, 100 μg of estradiol and 500 μg of dydrogesterone per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0063] Accurately measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, separation degree, etc. After comparison with the standard, the results of each peak are shown in Table 4:

[0064] Table 4

[0065] .

[0066] It can be seen from the results in the above table that this analytical method can effectively detect the impurities of estradiol under the detection condition of 220nm; and can effectively detect the impurities of dydrogesterone under the detection condition of 310nm.

[0067] Example 2 Determination of Related Substances in Estradiol Dydrogesterone Tablets

[0068] Chromatographic conditions:

[0069] Chromatographic column: ChromCore 120 C18 5μm, 4.6*150mm

[0070] Mobile phase: Mobile phase A is water; Mobile phase B is acetonitrile-methanol (1:1); Mobile phase C is ethanol;

[0071] Gradient elution:

[0072] .

[0073] Impurity mixed solution: Weigh appropriate amounts of 6-keto-estradiol, 17α-estradiol, estrone and dydrogesterone impurity A, 6-dehydroprogesterone, 17α-dydrogesterone and estradiol dydrogesterone tablet powder respectively, dilute with solvent (acetonitrile-water (40:60)) to prepare a solution containing approximately 0.5 μg of each impurity, 100 μg of estradiol and 500 μg of dydrogesterone per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0074] Accurately measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, separation degree, etc. After comparison with the standard, the results of each peak are shown in Table 5:

[0075] Table 5

[0076] .

[0077] It can be seen from the results in the above table that this analytical method can effectively detect the impurities of estradiol under the detection condition of 220nm; and can effectively detect the impurities of dydrogesterone under the detection condition of 310nm.

[0078] Example 3 Determination of Related Substances in Estradiol Dydrogesterone Tablets

[0079] Chromatographic conditions:

[0080] Column: thermo Hypersil ODS, 150×4.6 mm, 3 μm

[0081] Mobile phase: Mobile phase A is water; Mobile phase B is acetonitrile-methanol (1:1); Mobile phase C is ethanol;

[0082] Gradient elution:

[0083] .

[0084] Impurity mixed solution: Weigh appropriate amounts of 6-keto-estradiol, 17α-estradiol, estrone and dydrogesterone impurity A, 6-dehydroprogesterone, 17α-dydrogesterone and estradiol dydrogesterone tablet powder respectively, dilute with solvent (acetonitrile-water (40:60)) to prepare a solution containing approximately 0.5 μg of 6-keto-estradiol, 0.3 μg of 17α-estradiol, 0.5 μg of estrone, 0.5 μg of dydrogesterone impurity A, 0.13 μg of 6-dehydroprogesterone, 0.15 μg of 17α-dydrogesterone, 100 μg of estradiol and 500 μg of dydrogesterone per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0085] Accurately measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, separation degree, etc. After comparison with the standard, the results of each peak are shown in Table 6:

[0086] Table 6

[0087] .

[0088] It can be seen from the results in the above table that this analytical method can effectively detect the impurities of estradiol under the detection condition of 220nm; and can effectively detect the impurities of dydrogesterone under the detection condition of 310nm.

[0089] Example 4 Preliminary test on the accuracy of the method for related substances of estradiol dydrogesterone tablets

[0090] Take 5 estradiol tablets (22240506) respectively, put them in a 100ml volumetric bottle, add an appropriate amount of solvent (acetonitrile-water (40:60)), ultrasonically dissolve the estradiol tablets for 15 minutes, cool, add an appropriate amount of impurity mixed solution to the volumetric bottle, dilute to the scale with solvent, shake well, centrifuge at 2500rpm for 10min, and then filter with a 0.45μm polytetrafluoroethylene filter membrane, and take the filtrate as the series accuracy test solution. Under the proposed chromatographic conditions (same as Example 1), take the test solution for sample injection analysis, and use the external standard method to calculate the detection amount of each substance (estradiol-related impurities use estradiol as an external standard, and dydrogesterone-related impurities use dydrogesterone as an external standard), and use the ratio of the measured result (detection amount) to the theoretical value to calculate the recovery rate. The results are shown in Table 7.

[0091] Table 7

[0092] .

[0093] Relative correction factors for estradiol-related impurities:

[0094] Table 8

[0095]

[0096] Relative correction factors for dydrogesterone-related impurities:

[0097] Table 9

[0098] .

[0100] Conclusion: The recovery rates of all impurities of estradiol and dydrogesterone were acceptable within the concentration range of 20%~150% (acceptable standard 90.0%~108.0%).

[0101] Example 5 Investigation of the detection limits and quantitative items of related substances in estradiol dydrogesterone tablets

[0102] Take estradiol, dydrogesterone and related impurities, dissolve them in solvents and dilute them step by step, and inject and analyze the diluted solutions under the proposed chromatographic conditions (same as in Example 1), record the chromatogram, establish the quantitative limit according to the signal-to-noise ratio S / N≈10 / 1, and establish the detection limit according to the signal-to-noise ratio S / N≈3 / 1. The results are shown in Table 10:

[0103] Table 10 Results of detection limit and quantification limit tests

[0104]

[0105] Note: Test sample concentration: 0.1 mg / ml for estradiol and related impurities; 0.5 mg / ml for dydrogesterone and related impurities.

[0106] Conclusion: The detection limit and quantification limit of each component are lower than 0.05% of the test sample concentration. All impurities can be effectively detected and accurately quantified, and the method has good sensitivity.

[0107] Comparative Example 1: Determination of Related Substances in Estradiol Dydrogesterone Tablets

[0108] Test method: Determined according to the import registration standard JX20130310 method, the mobile phase is acetonitrile-water (425:600); isocratic elution; flow rate is 1.0ml / min; chromatographic column: thermo Hypersil ODS, 150×4.6mm, 3μm; column temperature is 40℃; injection volume is 20μl; running time is 40 minutes; detection wavelength is 220nm (estradiol and related impurities), 310nm (dydrogesterone and related impurities). The solvent used for diluting the sample is acetonitrile-water (40:60).

[0109] Impurity mixed solution: Weigh appropriate amounts of 6-keto-estradiol, 17α-estradiol, estrone and dydrogesterone impurity A, 6-dehydroprogesterone, 17α-dydrogesterone and estradiol dydrogesterone tablet powder respectively, dilute with solvent to prepare a solution containing approximately 0.5 μg of each impurity, 100 μg of estradiol and 500 μg of dydrogesterone per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0110] Accurately measure 20 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, separation degree, etc. After comparison with the standard, the results of each peak are shown in Table 11:

[0111] Table 11

[0112] .

[0113] The results showed that the method had interference in the detection of 6-keto-estradiol, poor separation of estradiol from adjacent impurities, and inability to effectively separate dydrogesterone from dydrogesterone impurity A, and 6-dehydroprogesterone from 17α-dydrogesterone.

[0114] Comparative Example 2: Determination of Related Substances in Estradiol Dydrogesterone Tablets

[0115] Test method: refer to the European Pharmacopoeia estradiol related substances method, the mobile phase is acetonitrile-ethanol (96)-water (21:25:54); isocratic elution; flow rate is 1.0ml / min; chromatographic column: Phenomenex Luna® C18(2) 100A, 4.6mm*25cm, 5μm; column temperature is 40℃; injection volume is 20μl; running time is 60 minutes; detection wavelength is 280nm. The solvent used for diluting the sample is acetonitrile-water (40:60).

[0116] Impurity mixed solution: Weigh appropriate amounts of 6-keto-estradiol, 17α-estradiol, estrone and dydrogesterone impurity A, 6-dehydroprogesterone, 17α-dydrogesterone and estradiol dydrogesterone tablet powder respectively, dilute with solvent to prepare a solution containing approximately 0.5 μg of each impurity, 100 μg of estradiol and 500 μg of dydrogesterone per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0117] Accurately measure 20 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, separation degree, etc. After comparison with the standard, the results of each peak are shown in Table 12:

[0118] Table 12

[0119] .

[0120] The results showed that the response value of 6-keto-estradiol was low under this method, and it was impossible to effectively separate dydrogesterone and dydrogesterone impurity A, and 6-dehydroprogesterone and 17α-dydrogesterone.

[0121] Comparative Example 3: Determination of Related Substances in Estradiol Dydrogesterone Tablets

[0122] Test method: refer to the United States Pharmacopoeia dydrogesterone related substances method, the mobile phase is acetonitrile-methanol-water (400:50:550); isocratic elution; flow rate is 1.0ml / min; chromatographic column: thermo Hypersil ODS, 150×4.6mm, 3μm; column temperature is 40℃; injection volume is 20μl; running time is 60 minutes; detection wavelength is 280nm.

[0123] Impurity mixed solution: Weigh appropriate amounts of 6-keto-estradiol, 17α-estradiol, estrone and dydrogesterone impurity A, 6-dehydroprogesterone, 17α-dydrogesterone and estradiol dydrogesterone tablet powder respectively, dilute with solvent to prepare a solution containing approximately 0.5 μg of 6-keto-estradiol, 0.3 μg of 17α-estradiol, 0.5 μg of estrone, 0.5 μg of dydrogesterone impurity A, 0.13 μg of 6-dehydroprogesterone, 0.15 μg of 17α-dydrogesterone, 100 μg of estradiol and 500 μg of dydrogesterone per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0124] Accurately measure 20 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, separation degree, etc. After comparison with the standard, the results of each peak are shown in Table 13:

[0125] Table 13

[0126] .

[0127] The results showed that the response values ​​of the impurities of estradiol were low, the separation of estrone and 17α-estradiol was poor, and it was impossible to effectively separate dydrogesterone and dydrogesterone impurity A under this method.

[0128] Comparative Example 4 Determination of Related Substances in Estradiol Dydrogesterone Tablets

[0129] Chromatographic conditions:

[0130] Column: thermo Hypersil ODS, 150×4.6 mm, 3 μm

[0131] Mobile phase: Mobile phase A is water; Mobile phase B is acetonitrile; Mobile phase C is ethanol;

[0132] Gradient elution:

[0133]

[0134] Impurity mixed solution: Weigh appropriate amounts of 6-keto-estradiol, 17α-estradiol, estrone and dydrogesterone impurity A, 6-dehydroprogesterone, 17α-dydrogesterone and estradiol dydrogesterone tablet powder respectively, dilute with solvent to prepare a solution containing approximately 0.5 μg of 6-keto-estradiol, 0.3 μg of 17α-estradiol, 0.5 μg of estrone, 0.5 μg of dydrogesterone impurity A, 0.13 μg of 6-dehydroprogesterone, 0.15 μg of 17α-dydrogesterone, 100 μg of estradiol and 500 μg of dydrogesterone per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0135] Accurately measure 20 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, separation degree, etc. After comparison with the standard, the results of each peak are shown in Table 14:

[0136] Table 14

[0137] .

[0138] It can be seen from the results in the above table that the analytical method can effectively detect the various impurities of estradiol under the detection condition of 220nm, but the baseline fluctuates greatly and the estrone integral is easily interfered; under the detection condition of 310nm, the dydrogesterone impurity A completely overlaps with the dydrogesterone peak and cannot be separated.

[0139] Comparative Example 5 Determination of Related Substances in Estradiol Dydrogesterone Tablets

[0140] Chromatographic conditions:

[0141] Column: thermo Hypersil ODS, 150×4.6 mm, 3 μm

[0142] Mobile phase: Mobile phase A is water; Mobile phase B is acetonitrile-methanol (4:1); Mobile phase C is ethanol;

[0143] Gradient elution:

[0144]

[0145] Impurity mixed solution: Weigh appropriate amounts of 6-keto-estradiol, 17α-estradiol, estrone and dydrogesterone impurity A, 6-dehydroprogesterone, 17α-dydrogesterone and estradiol dydrogesterone tablet powder respectively, dilute with solvent to prepare a solution containing approximately 0.5 μg of 6-keto-estradiol, 0.3 μg of 17α-estradiol, 0.5 μg of estrone, 0.5 μg of dydrogesterone impurity A, 0.13 μg of 6-dehydroprogesterone, 0.15 μg of 17α-dydrogesterone, 100 μg of estradiol and 500 μg of dydrogesterone per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0146] Accurately measure 20 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, separation degree, etc. After comparison with the standard, the results of each peak are shown in Table 15:

[0147] Table 15

[0148] .

[0149] It can be seen from the results in the above table that the analytical method can effectively detect the various impurities of estradiol under the detection condition of 220nm, but the baseline fluctuates greatly, and the integrals of 17α-estradiol and estrone are easily interfered; under the detection condition of 310nm, the peaks of dydrogesterone impurity A and dydrogesterone are almost completely overlapped and cannot be separated.

Claims

1. A method for detecting and separating estradiol and / or dydrogesterone, characterized in that: It includes the following steps: Liquid chromatography is used to detect or separate the sample to be tested; the sample to be tested includes substance 1 and / or substance 2, the substance 1 is estradiol and impurity 1, the impurity 1 is selected from one or more of 17α-estradiol, estrone, Δ9,11-estradiol and 6-keto-estradiol, the substance 2 is dydrogesterone and impurity 2, the impurity 2 is selected from one or more of dydrogesterone impurity A, 6-dehydroprogesterone and 17α-dydrogesterone; Wherein, the detection conditions of the liquid chromatography method include: The chromatographic column is an octadecyl bonded silica reverse phase chromatographic column; Mobile phase A was water; Mobile phase B was acetonitrile-methanol; Mobile phase C was 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 procedure is as follows: ； Among them, X1 is 62-64%; X2 is 50-52%; Y1 is 36-38%; Y2 is 10%; and Y3 is 35%.

2. The detection and separation method according to claim 1, characterized in that: The elution program of the linear gradient elution is: or, 。 3. The detection and separation method according to claim 1, characterized in that: It meets one or more of the following conditions: (1) The impurity 1 is selected from one or more of 17α-estradiol, estrone and 6-keto-estradiol; (2) The chromatographic column is ChromCore 120 C18 or thermo Hypersil ODS; (3) The filler particle size of the chromatographic column is 2.5-7.5 μm; (4) The length of the chromatographic column is 100-200 mm; (5) The inner diameter of the chromatographic column is 3-5 mm; (6) The column temperature of the chromatographic column is 35-45°C; (7) The flow rate of the mobile phase is 0.8-1.2 mL / min; (8) The injection volume of the liquid chromatography method is 20-80 μL.

4. The detection and separation method according to claim 3, characterized in that: It meets one or more of the following conditions: (1) The filler particle size of the chromatographic column is 5 μm or 3 μm; (2) The length of the chromatographic column is 150 mm; (3) The inner diameter of the chromatographic column is 4.6 mm; (4) The column temperature of the chromatographic column is 40°C; (5) The flow rate of the mobile phase is 1.0 mL / min; (6) The injection volume of the liquid chromatography method is 50 μL.

5. The detection and separation method according to claim 3, characterized in that: The chromatographic column is ChromCore 120 C18 4.6*150 mm, 5 μm or thermo Hypersil ODS, 150×4.6 mm, 3 μm.

6. The detection and separation method according to claim 1, characterized in that: The detector of the detection and separation method is an ultraviolet detector; Preferably, when the sample to be tested contains substance 1, the detection wavelength of the detector is 220 nm; And / or, when the sample to be tested contains substance 2, the detection wavelength of the detector is 310 nm.

7. The detection and separation method according to claim 1, characterized in that: The method comprises the following steps: before testing, pre-treating the sample to be tested; The pretreatment comprises: dissolving the sample to be tested in a diluent; the diluent is a mixed solution of acetonitrile and water; preferably, the volume ratio of acetonitrile to water is 40:

60.

8. The detection and separation method according to claim 7, characterized in that: The mass volume ratio of each substance in the sample to be tested to the diluent is 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.

9. The detection and separation method according to claim 8, characterized in that: The method comprises the following steps: after the dissolving step, a filtering step is further included, for example, filtering is performed using a polytetrafluoroethylene microporous filter membrane.

10. The detection and separation method according to claim 1, characterized in that: The sample to be tested consists of 17α-estradiol, estrone, estradiol, 6-keto-estradiol, dydrogesterone, dydrogesterone impurity A, 6-dehydroprogesterone and 17α-dydrogesterone.

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