Detection method of estradiol valerate tablet related substances

Abstract

The invention belongs to the field of medicine detection, and relates to an estradiol valerate tablet related substance detection method, which comprises: detecting a mixed solution containing estradiol valerate and impurities by using a high performance liquid chromatography; the impurities comprise one or more of impurities A, G, K, L, C, D and E; a chromatographic column adopted by the high performance liquid chromatography is a column which takes phenyl hexyl bonded silica gel with the particle size of 3 microns as a filling agent; a mobile phase A and a mobile phase B are adopted in the high performance liquid chromatography; wherein the mobile phase A is water, and the mobile phase B is acetonitrile; the detection method for the estradiol valerate tablet related substances, provided by the invention, has the advantages of simple detection, few used samples and more reliable and more accurate analysis and detection results.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a method for detecting related substances in estradiol valerate tablets. Background Art

[0002] Estradiol valerate is a steroid developed by Endo Pharmaceuticals and is an ERs agonist. Currently, the drug is in the highest development stage for approval and is used to treat estrogen deficiency syndrome and endocrine system diseases. The molecular formula of estradiol valerate is C 23 H 32 O3, molecular weight is 356.50, chemical name is Estra-1,3,5(10)-triene-3,17-diol(17b)-,17-pentanoate, chemical structure information is as follows:

[0003]

[0004] Impurities in estradiol valerate tablets primarily originate from the introduction of the active pharmaceutical ingredient, the production process, and storage. Therefore, developing a method to effectively isolate impurities in estradiol valerate tablets is crucial for quality control.

[0005] Based on the impurity spectrum of the estradiol valerate API manufacturer and the impurities in the import registration standard for estradiol valerate, it is planned to study the six impurities listed in Table 1 in estradiol valerate tablets.

[0006] Table 1: List of impurities in estradiol valerate tablets

[0007]

[0008]

[0009] The relevant substance method in the import registration standard for estradiol valerate tablets (JX20100066) is isocratic elution, which cannot effectively separate the above-mentioned impurities. In addition, it is necessary to use the external standard method of two main components (estradiol valerate and levonorgestrel) at dual wavelengths (242nm and 280nm) for control. The detection is cumbersome and wastes reference materials.

[0010] In the prior art, patent document CN 117871705 A discloses a method for analyzing related substances in estradiol valerate tablets. However, in the specificity test of Example 1 of the document, the separation degree of impurities A and C is na, which cannot be effectively evaluated and the analytical test results are unreliable. In addition, the separation degree of impurity L 6-ketoestradiol valerate in Example 1 of the document is low. Summary of the Invention

[0011] The present invention aims to provide a method for detecting related substances in estradiol valerate tablets, which is simple to detect, requires less sample, and provides more reliable and accurate analysis and detection results.

[0012] In order to solve the above technical problems, the present invention provides a method for detecting related substances in estradiol valerate tablets as follows:

[0013] A method for detecting related substances in estradiol valerate tablets comprises the following steps:

[0014] High performance liquid chromatography is used to detect a mixed solution, wherein the mixed solution contains estradiol valerate and impurities; the impurities contain one or more of impurities A, G, K, L, C, D, and E;

[0015] The chromatographic column used in the high performance liquid chromatography method is a column filled with phenylhexyl bonded silica gel with a particle size of 3 μm;

[0016] The high performance liquid chromatography method uses mobile phase A and mobile phase B; wherein the mobile phase A is water and the mobile phase B is acetonitrile;

[0017] The high performance liquid chromatography method adopts gradient elution; the gradient elution conditions are:

[0018]

[0019] Optionally, the chromatographic column is 4.6 mm×250 mm, 3 μm phenylhexyl bonded silica gel.

[0020] Optionally, the chromatographic column is Phenyl-Hexyl, 4.6 mm × 250 mm, 3 μm phenyl-hexyl bonded silica gel.

[0021] Optionally, the HPLC conditions are: flow rate of 0.65 ml / min to 0.75 ml / min; column temperature of 28° C. to 32° C.; sample plate temperature of 10° C.; detection wavelength of 220 nm; and injection volume of 10 μl to 30 μl.

[0022] Optionally, the HPLC conditions are: flow rate of 0.7 ml / min; column temperature of 30° C.; sample plate temperature of 10° C.; detection wavelength of 220 nm; and injection volume of 20 μl.

[0023] Optionally, the solvents of the mixed solution are: acetonitrile, methanol and water, and the volume ratio of the acetonitrile, methanol and water is: 60:10:30.

[0024] Optionally, the content of the impurity is calculated according to the principal component external standard method with the addition of a correction factor.

[0025] Alternatively, the mixed solution is prepared as follows: 20 estradiol valerate tablets are placed in a 20 ml volumetric flask, 10 ml of solvent is added, and the mixture is heated and shaken in a 50°C water bath for 10 minutes to disintegrate, followed by ultrasonication for 15 minutes to dissolve the estradiol valerate, and cooled. 1 ml each of impurity A reference stock solution, 6α-OH-estradiol valerate reference stock solution, 6-ketoestradiol valerate reference stock solution, impurity G reference stock solution, impurity C reference stock solution, and impurity E reference stock solution are added, diluted to the mark with solvent, and shaken well. An appropriate amount is taken, centrifuged, the supernatant is taken, filtered, and the filtrate is taken to obtain the solution.

[0026] Optionally, the concentration of estradiol valerate in the mixed solution is 0.8-1.2 mg / ml.

[0027] Optionally, the impurity content in the impurity reference stock solution is 0.8-1.2 mg / ml.

[0028] The method for detecting related substances in estradiol valerate tablets provided by the present invention adopts gradient elution, which saves more control samples and is simpler than the prior art. In the specificity test of Example 1 of the present invention, the separation degree of impurity A of the present invention cannot be effectively evaluated relative to the separation degree of the prior art, and the separation degree of impurity A of the present invention is relatively large. In addition, the separation degree of impurity L in Example 1 is also much greater than the separation degree of impurity L in the prior art. This shows that the detection results of the present invention are more reliable and accurate.

[0029] In Example 2 of the present invention, the detection limit and the quantification limit were tested. Under the condition that the signal-to-noise ratio was not less than 3, the detection limit was very low; under the condition that the average signal-to-noise ratio was greater than 10, the quantification limit was also not high, indicating that the detection method of the present invention has high sensitivity.

[0030] The linearity and range tests of Example 3 of the present invention showed that the peak area and concentration had a good linear relationship within the range of the quantitative limit solution concentration to 150% of the limit concentration, and the main component external standard method with the correction factor could be used for determination.

[0031] In Example 4 of the present invention, an external standard method for the principal component of the correction factor was tested, and a linear operation test was performed by different personnel to obtain the results. The difference between the test results of different personnel for each impurity and the test results of different personnel was less than or equal to 0.02, and the difference between impurity A, impurity L and impurity E was 0, indicating that the detection method of the present invention has good repeatability.

[0032] The intermediate precision test results showed that: the same sample was taken at different times and by different personnel, and each measurement was performed 6 times. The absolute differences between the maximum and minimum values of the impurity A, 6α-OH-estradiol valerate, 6-ketoestradiol valerate, impurity G, impurity C, impurity E, other single impurities, and total impurities in the 12 spiked test samples all met the requirements, indicating that the detection method of the present invention has good intermediate precision.

[0033] In the control test of the present invention, the related substance method in the import registration standard (JX20100066) of estradiol valerate tablets was used. Baseline separation could not be achieved between impurity G and impurity C, and between impurity C and estradiol valerate peaks in the mixed solution. This method is not suitable for the control of the six impurities in this product.

[0034] Compared with the analysis method for related substances in estradiol valerate tablets disclosed in patent document CN 117871705 A, the present invention adopts different chromatographic columns, different mobile phase A, different gradient elution time, and different ratios of mobile phases A and B. The separation degree of impurities A and impurity L in the present invention is higher, and the detection method is more reliable and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the blank solution chromatogram in the specificity of Example 1;

[0036] Figure 2 This is the chromatogram of the blank excipient solution in the specificity of Example 1;

[0037] Figure 3 This is a chromatogram of the solution for locating impurity A in the specificity of Example 1;

[0038] Figure 4 This is a chromatogram of the solution for locating impurity C in the specificity of Example 1;

[0039] Figure 5 This is a chromatogram of the solution for locating impurity E in the specificity of Example 1;

[0040] Figure 6 This is a chromatogram of the solution for locating the impurity 6α-OH-estradiol valerate in the specificity of Example 1;

[0041] Figure 7 This is a chromatogram of the solution for locating the impurity 6-ketovalerate estradiol in the specificity of Example 1;

[0042] Figure 8 This is a chromatogram of the solution for localizing impurity G (Δ6-estradiol valerate) in the specificity of Example 1;

[0043] Figure 9 This is a chromatogram of the mixed solution in the specificity of Example 1;

[0044] Figure 10 This is the chromatogram of the test solution in Example 1;

[0045] Figure 11 The chromatogram is the detection limit of Example 2;

[0046] Figure 12 The chromatogram is the quantitative limit of Example 2;

[0047] Figure 13 This is the chromatogram of the mixed solution in Comparative Example 1 at a wavelength of 280 nm;

[0048] Figure 14 This is the chromatogram of the mixed solution in Comparative Example 1 at a wavelength of 242 nm. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail in the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0050] The invention provides a method for detecting related substances of estradiol valerate tablets. The method adopts high performance liquid chromatography to detect the related substances of estradiol valerate tablets, and adopts a principal component external standard method with a correction factor to calculate the impurity content.

[0051] In the present invention, the related substances are impurity A (estra-1,3,5(10)-triene-3,17β-diol), impurity C (C 23 H 30 O3), impurities E(C 28 H 40 O4), impurity K (6α-OH-estradiol valerate), impurity L (6-ketoestradiol valerate), impurity G (△ 6-estradiol valerate).

[0052] In the present invention, the chromatographic column preferably uses phenylhexyl bonded silica gel with a particle size of 3 μm as a filler.

[0053] In the present invention, the chromatographic column model is preferably Phenyl-Hexyl, 4.6mm×250mm, 3μm.

[0054] In the present invention, the flow rate is 0.65 ml / min to 0.75 ml / min; the column temperature is 28° C. to 32° C.; the sample plate temperature is 10° C.; the detection wavelength is 220 nm; and the injection volume is 10 μl to 30 μl.

[0055] In the present invention, the flow rate is preferably 0.7 ml / min; the column temperature is preferably 30° C.; and the injection volume is preferably 20 μl.

[0056] In the present invention, mobile phase A is water, mobile phase B is acetonitrile, and gradient elution is performed.

[0057] In the present invention, the gradient elution procedure is as follows:

[0058]

[0059]

[0060] In the present invention, the gradient elution procedure is as follows: the mixed solution is estradiol valerate and impurity A (estra-1,3,5(10)-triene-3,17β-diol), impurity C (C 23 H 30 O3), impurities E(C 28 H 40 O4), impurity K (6α-OH-estradiol valerate), impurity L (6-ketoestradiol valerate), and impurity G (△6-estradiol valerate).

[0061] Example 1 Detection of Related Substances in Estradiol Valerate Tablets - Specificity

[0062] 1. The reagents, reference substances and sample information used in the examples of the present invention are as follows:

[0063] Table 2 Reagents, reference substances and sample information:

[0064]

[0065] 2. Solution preparation

[0066] Solvent: acetonitrile-methanol-water (60:10:30)

[0067] Excipient solution: Place approximately 1460 mg of blank excipient in a 10 ml volumetric flask, add 8 ml of solvent, heat and shake in a 50°C water bath for 10 minutes, then sonicate for 15 minutes. Allow to cool, dilute to volume with solvent, and shake well. Take an appropriate amount, centrifuge, remove the supernatant, filter, and obtain the filtrate.

[0068] Impurity reference stock solution: Take an appropriate amount of each impurity, dissolve it with solvent and dilute it to make a solution of about 0.1 mg per 1 ml, which will be used as the reference stock solution of each impurity.

[0069] Localization solution for each impurity: Take an appropriate amount of the stock solution of each impurity reference substance and dilute it with solvent to make a solution containing approximately 5 μg per 1 ml.

[0070] Reference substance solution: Take an appropriate amount of estradiol valerate reference substance, accurately weigh it, dissolve it in solvent and dilute it to make a solution containing approximately 0.1 mg per 1 ml as the stock solution; accurately measure an appropriate amount of the stock solution, dilute it with solvent to make a solution containing approximately 0.01 mg of estradiol valerate per 1 ml, shake it well, filter it, and take the filtrate to obtain the solution.

[0071] Test solution: Place 10 tablets of this product in a 10ml volumetric flask, add 8ml of solvent, heat and shake in a 50°C water bath for 10 minutes to disintegrate, then sonicate for 15 minutes to dissolve the estradiol valerate. Allow to cool, dilute to the mark with solvent, and shake well. Take an appropriate amount, centrifuge, remove the supernatant, filter, and obtain the filtrate.

[0072] Mixed solution: Take 20 tablets of this product and place them in a 20ml volumetric flask. Add 10ml of solvent and heat and shake in a 50℃ water bath for 10 minutes to disintegrate. Then, sonicate for 15 minutes to dissolve the estradiol valerate. Let cool. Add 1ml each of the Impurity A Reference Stock Solution, 6α-OH-Estradiol Valerate Reference Stock Solution, 6-Ketoestradiol Valerate Reference Stock Solution, Impurity G Reference Stock Solution, Impurity C Reference Stock Solution, and Impurity E Reference Stock Solution. Dilute to the mark with solvent and shake well. Take an appropriate amount, centrifuge, remove the supernatant, filter, and obtain the filtrate.

[0073] 3. HPLC conditions:

[0074] Instrument: Thermo high performance liquid chromatograph;

[0075] Chromatographic column: phenylhexyl bonded silica gel as filler ( Phenyl-Hexyl, 4.6 mm × 250 mm, 3 μm or equivalent performance column)

[0076] Flow rate: 0.7 ml / min;

[0077] Column temperature: 30°C;

[0078] Detection wavelength: 220nm;

[0079] Injection volume: 20 μl;

[0080] Mobile phase: Water is mobile phase A, acetonitrile is mobile phase B, and the gradient elution program is as follows:

[0081]

[0082]

[0083] According to the above chromatographic conditions, 20 μl of blank excipient solution, impurity location solution, mixed solution and test solution were accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The specificity results are shown in Table 3 and Figures 1 to 10 .

[0084] Table 3 Specificity results

[0085]

[0086] The results showed that the blank excipients did not interfere with the determination of related substances; the resolution between the main peak and adjacent peaks, as well as between known impurity peaks, met the requirements. Impurity E had no peak, so there was no resolution.

[0087] Resolution is a measure of the degree of separation between two adjacent chromatographic peaks. It is calculated as Rs = 2(tR2 - tR1) / (W1 + W2), where tR is the retention time and W is the peak width. If the two peaks completely overlap, the resolution may be close to zero or impossible to calculate.

[0088] Example 2 Detection limit and quantification limit

[0089] The HPLC chromatographic conditions were the same as those in Example 1.

[0090] Take appropriate amounts of each impurity location solution and reference solution prepared in Example 1 and gradually dilute them to prepare a mixed solution. The signal-to-noise ratio (S / N) of each component in the detection limit solution is not less than 3; the signal-to-noise ratio (S / N) of each component in the quantification limit solution is not less than 10.

[0091] According to the chromatographic conditions described in Example 1, 20 μl was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 4, Figures 11-12 .

[0092] Table 4:

[0093]

[0094] The results showed that the detection limits of impurity A, 6α-OH-estradiol valerate, 6-ketoestradiol valerate, impurity G, impurity C, impurity E and estradiol valerate were 0.57 ng, 1.55 ng, 0.98 ng, 1.59 ng, 2.17 ng, 1.53 ng, 4.67 ng and 1.42 ng, respectively, and the signal-to-noise ratios were not less than 3; the quantification limits of impurity A, 6α-OH-estradiol valerate, 6-ketoestradiol valerate, impurity G, impurity C, impurity E and estradiol valerate were 1.89 ng, 5.17 ng, 3.26 ng, 5.30 ng, 7.22 ng, 5.09 ng, 15.56 ng and 4.73 ng, respectively, and the signal-to-noise ratios were not less than 10; the method had high sensitivity.

[0095] Example 3 Linearity and range test

[0096] The HPLC chromatographic conditions were the same as those in Example 1.

[0097] Quantitation limit solution: Take the quantitation limit solution prepared in Example 2.

[0098] Linear stock solution: Take 5 ml each of the reference substance stock solution and impurity A reference substance stock solution, 6α-OH-estradiol valerate reference substance stock solution, 6-ketoestradiol valerate reference substance stock solution, impurity G reference substance stock solution, impurity C reference substance stock solution and impurity E reference substance stock solution, place them in a 50 ml volumetric flask, dilute to the scale with solvent, shake well to obtain 200% linear solution.

[0099] Accurately measure 2.5 ml, 4 ml, 5 ml, and 7.5 ml of the linear stock solution and place them in different 10 ml volumetric flasks. Dilute to the scale with solvent and shake well to prepare 50%, 80%, 100%, and 150% linear solutions, respectively.

[0100] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram.

[0101] Linear investigation results

[0102] Table 5: Estradiol valerate linearity and range determination results

[0103]

[0104] Table 6: Impurity A linearity and range determination results

[0105]

[0106]

[0107] Table 7: 6α-OH-estradiol valerate linearity and range determination results

[0108]

[0109] Table 8: 6-Ketovalerate Estradiol Linearity and Range Determination Results

[0110]

[0111]

[0112] Table 9: Impurity G linearity and range determination results

[0113]

[0114] Table 10: Impurity C linearity and range determination results

[0115]

[0116]

[0117] Table 11: Impurity E linearity and range determination results

[0118]

[0119] The results showed that when the concentration of each impurity A was 0.0947μg / ml~9.4721μg / ml, the peak area had a good linear relationship with the concentration, the linear equation was y=0.8528x-0.0725, and the correlation coefficient r was 0.9995;

[0120] When the concentration of 6α-OH-estradiol valerate was 0.2586μg / ml~10.3438μg / ml, the peak area had a good linear relationship with the concentration, the linear equation was y=0.6628x-0.0672, and the correlation coefficient r was 0.9994;

[0121] When the concentration of 6-ketovalerate estradiol was 0.1632μg / ml~10.8783μg / ml, the peak area had a good linear relationship with the concentration, the linear equation was y=1.4817x-0.1575, and the correlation coefficient r was 0.9994;

[0122] When the concentration of impurity G is 0.2651μg / ml~10.6046μg / ml, the peak area has a good linear relationship with the concentration. The linear equation is y=2.0461x-0.1990, and the correlation coefficient r is 0.9995.

[0123] When the concentration of impurity C is 0.3609μg / ml~9.6236μg / ml, the peak area has a good linear relationship with the concentration. The linear equation is y=1.4406x-0.1431, and the correlation coefficient r is 0.9995.

[0124] When the concentration of impurity E is 0.2367μg / ml~9.4690μg / ml, the peak area has a good linear relationship with the concentration. The linear equation is y=0.6139x+0.0088, and the correlation coefficient r is 0.9989.

[0125] When the concentration of estradiol valerate was 0.7779 μg / ml~10.3720 μg / ml, the peak area had a good linear relationship with the concentration, the linear equation was y=0.6679x-0.1442, and the correlation coefficient r was 0.9986.

[0126] Example 4 Correction Factor Main Component External Standard Method Test

[0127] The correction factor is calculated based on the linear results of different personnel and is calculated as the average value. The results of one experimenter are shown in Example 3.

[0128] The HPLC chromatographic conditions were the same as those in Example 1.

[0129] The quantitative solutions and linear solutions for different personnel were prepared in the same manner as in Example 3.

[0130] Accurately measure 20 μl of each linear solution, inject it into the liquid chromatograph, and record the chromatographic results. See Table 12 below.

[0131] Table 12 Correction factor data

[0132] Components Average correction factor Impurity A 0.78 6α-OH estradiol valerate 1.01 Estradiol 6-ketovalerate 0.45 Impurity G 0.32 Impurity C 0.46 Impurity E 1.09

[0133] 1. Precision

[0134] The HPLC chromatographic conditions were the same as those in Example 1.

[0135] Reference substance solution: Take an appropriate amount of estradiol valerate reference substance, accurately weigh it, dissolve it in solvent and dilute it to make a solution containing approximately 0.1 mg per 1 ml as the stock solution; accurately measure an appropriate amount of the stock solution, dilute it with solvent to make a solution containing approximately 0.01 mg of estradiol valerate per 1 ml, shake it well, filter it, and take the filtrate to obtain the solution.

[0136] Stock solution of impurity reference substances: Accurately weigh an appropriate amount of each impurity reference substance and dilute it with solvent to prepare a mixed solution containing approximately 50 μg each of impurity A, 6α-OH estradiol valerate, 6-ketoestradiol valerate, impurity G, impurity C, and impurity E per 1 ml.

[0137] For spiked test solution: Place 10 tablets of this product in a 10ml volumetric flask, add 8ml of solvent, and heat and shake in a 50°C water bath for 10 minutes to disintegrate. Then, sonicate for 15 minutes to dissolve the estradiol valerate. Allow to cool, accurately add 1ml of the impurity reference stock solution, dilute to the mark with solvent, and shake well. Take an appropriate amount, centrifuge, remove the supernatant, filter, and obtain the filtrate. Prepare 6 replicates.

[0138] Accurately measure 20 μl of the spiked test solution and reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate according to the external standard method of adding the correction factor principal component. The results are shown in Table 13.

[0139] Table 13 Repeatability results

[0140]

[0141] The results showed that the differences between the maximum and minimum values of 6 samples met the requirements and the method had good repeatability.

[0142] 2. Intermediate precision

[0143] Different experimenters conducted intermediate precision tests using liquid chromatography at different times, using the above-mentioned determination method. The results were compared with those of the repeatability test. The results are shown in Table 14 below.

[0144] Table 14 Intermediate precision test results

[0145]

[0146]

[0147] The results showed that the absolute differences between the maximum and minimum values of impurity A, 6α-OH-estradiol valerate, 6-ketoestradiol valerate, impurity G, impurity C, impurity E, other single impurities and total impurities in 12 spiked samples were in compliance with the requirements; the intermediate precision was good.

[0148] Example 5 Detection of Related Substances in Estradiol Valerate Tablets - Specificity

[0149] The instruments and reagents used in this example are the same as those in Example 1

[0150] The difference from Example 1 is that the conditions of HPLC are:

[0151] Instrument: Thermo high performance liquid chromatograph;

[0152] Chromatographic column: phenylhexyl bonded silica gel as filler ( Phenyl-Hexyl, 4.6mm×250mm, 3μm)

[0153] Flow rate: 0.65 ml / min;

[0154] Column temperature: 28°C;

[0155] Detection wavelength: 220nm;

[0156] Injection volume: 30 μl;

[0157] The specificity results are shown in Table 15 below:

[0158]

[0159] Example 6 Detection of Related Substances in Estradiol Valerate Tablets - Specificity

[0160] The instruments and reagents used in this example are the same as those in Example 1

[0161] The difference from Example 1 is that the conditions of HPLC are:

[0162] Instrument: Thermo high performance liquid chromatograph;

[0163] Chromatographic column: phenylhexyl bonded silica gel as filler ( Phenyl-Hexyl, 4.6mm×250mm, 3μm)

[0164] Flow rate: 0.75 ml / min;

[0165] Column temperature: 32°C;

[0166] Detection wavelength: 220nm;

[0167] Injection volume: 10 μl;

[0168] The specificity results are shown in Table 16 below:

[0169]

[0170]

[0171] Comparative Example 1-Estradiol Valerate Tablets Import Registration Standard (JX20100066)

[0172] 1. The reagents, reference substances and sample information used in Comparative Example 1 are as follows:

[0173] Table 17 Reagents, reference substances and sample information:

[0174]

[0175] 2. Solution preparation

[0176] Solvent: Measure 600ml of acetonitrile, 100ml of methanol and 300ml of water respectively and mix them evenly to obtain the product.

[0177] Blank excipient solution: Take about 790 mg of blank excipient, accurately weigh, place in a 10 ml volumetric flask, add 8 ml of solvent, heat and shake in a 50 ° C water bath for 10 minutes, then ultrasonic for 15 minutes, let cool, dilute to the scale with solvent, shake well, centrifuge, and take the supernatant.

[0178] Test solution: Take 10 tablets of this product, place them in a 10ml volumetric flask, add 8ml of solvent, heat and shake in a 50℃ water bath for 10 minutes to disintegrate, then ultrasonicate for 15 minutes to dissolve the estradiol valerate, cool, dilute to the scale with solvent, shake well, and centrifuge to obtain the solution.

[0179] Levonorgestrel reference substance stock solution: Take an appropriate amount of levonorgestrel reference substance, accurately weigh it, dissolve it in solvent and dilute it to make a solution containing approximately 0.75 mg per 1 ml.

[0180] Levonorgestrel reference substance stock solution ①: Accurately measure an appropriate amount of levonorgestrel reference substance stock solution and dilute it with solvent to make a solution containing approximately 7.5 μg per 1 ml.

[0181] Reference solution: Accurately weigh 10 mg of estradiol valerate working reference substance into a 10 ml volumetric flask, accurately add 1 ml of levonorgestrel reference substance stock solution, and dilute to the scale with solvent; accurately measure 1 ml and place it into a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0182] Take appropriate amounts of estradiol valerate, estradiol, 6α-OH-estradiol valerate, 6-ketoestradiol valerate, △6-estradiol valerate, impurity C, impurity D, and impurity E reference substances to prepare stock solutions with a concentration of approximately 50 μg and 5 μg / ml positioning solutions, respectively.

[0183] Levonorgestrel targeting solution: Accurately measure an appropriate amount of levonorgestrel reference substance stock solution ① and dilute it with solvent to make a solution containing approximately 0.75 μg per 1 ml.

[0184] Mixed solution: Take about 20 mg of estradiol valerate reference substance, accurately weigh it, and place it in a 20 ml volumetric flask. Add 2 ml each of levonorgestrel reference substance stock solution ①, estradiol reference substance stock solution, 6α-OH-estradiol valerate reference substance stock solution, 6-ketovalerate estradiol reference substance stock solution and △6-estradiol valerate reference substance stock solution, impurity C reference substance stock solution, impurity D reference substance stock solution and impurity E reference substance stock solution, dissolve it in solvent and dilute it to the scale. Mix well to obtain the solution.

[0185] 3. HPLC conditions:

[0186] Instrument: Thermo high performance liquid chromatograph;

[0187] Chromatographic column: Wasters Novapack Phenyl (150mm*3.9mm, 4μm)

[0188] Flow rate: 1.0 ml / min;

[0189] Column temperature: 30°C;

[0190] Detection wavelength: 280nm and 242nm;

[0191] Injection volume: 50 μl;

[0192] Mobile phase: water-acetonitrile (60:40), isocratic elution

[0193] According to the above chromatographic conditions, 50 μl of blank excipient solution, impurity location solution, mixed solution and test solution were accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 18, and the spectrum of the mixed solution is shown in Figure 13 .

[0194] Table 18 Comparative Example Results

[0195]

[0196] The results showed that the related substance method in the import registration standard for estradiol valerate tablets (JX20100066) could not achieve baseline separation between impurity G and impurity C, and between impurity C and estradiol valerate peaks in the mixed solution. This method is not suitable for the control of the six impurities in this product.

[0197] The present invention is compared with the analysis method for related substances in estradiol valerate tablets disclosed in patent document CN 117871705 A. The chromatographic columns used are different, the mobile phase A is different, the gradient elution time, and the ratio of mobile phases A and B are different. The high-performance liquid chromatography conditions of the present invention are also significantly different from those in the document. In addition, the separation degree of impurities A and L in the present invention is higher, and the detection method of the present invention is more reliable and accurate.

[0198] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting related substances in estradiol valerate tablets, characterized in that: The steps include: High performance liquid chromatography is used to detect a mixed solution, wherein the mixed solution contains estradiol valerate and impurities; the impurities contain one or more of impurities A, G, K, L, C, D, and E; The chromatographic column used in the high performance liquid chromatography method is a column filled with phenylhexyl bonded silica gel with a particle size of 3 μm; The high performance liquid chromatography method uses mobile phase A and mobile phase B; wherein the mobile phase A is water and the mobile phase B is acetonitrile; The high performance liquid chromatography method adopts gradient elution; the gradient elution conditions are:

2. The method for detecting related substances in estradiol valerate tablets according to claim 1, wherein The chromatographic column is 4.6 mm×250 mm, 3 μm phenylhexyl bonded silica gel.

3. The method for detecting related substances in estradiol valerate tablets according to claim 2, wherein The chromatographic column is Phenyl-Hexyl, 4.6 mm × 250 mm, 3 μm phenyl-hexyl bonded silica gel.

4. The method for detecting related substances in estradiol valerate tablets according to any one of claims 1 to 3, characterized in that: The high performance liquid chromatography conditions are: flow rate of 0.65 ml / min to 0.75 ml / min; column temperature of 28° C. to 32° C.; sample plate temperature of 10° C.; detection wavelength of 220 nm; and injection volume of 10 μl to 30 μl.

5. The method for detecting related substances in estradiol valerate tablets according to claim 4, wherein The HPLC conditions are as follows: flow rate of 0.7 ml / min; column temperature of 30° C.; sample plate temperature of 10° C.; detection wavelength of 220 nm; and injection volume of 20 μl.

6. The method for detecting related substances in estradiol valerate tablets according to claim 5, wherein: The solvents of the mixed solution are acetonitrile, methanol and water, and the volume ratio of the acetonitrile, methanol and water is 60:10:

30.

7. The method for detecting related substances in estradiol valerate tablets according to claim 6, wherein: The content of the impurity is calculated according to the principal component external standard method with the addition of a correction factor.

8. The method for detecting related substances in estradiol valerate tablets according to claim 7, wherein: The mixed solution is prepared as follows: 20 estradiol valerate tablets are placed in a 20ml volumetric flask, 10ml of solvent is added, and the mixture is heated and shaken in a 50°C water bath for 10 minutes to disintegrate. The mixture is then ultrasonicated for 15 minutes to dissolve the estradiol valerate. The mixture is cooled, and 1ml each of impurity A reference stock solution, 6α-OH-estradiol valerate reference stock solution, 6-ketoestradiol valerate reference stock solution, impurity G reference stock solution, impurity C reference stock solution, and impurity E reference stock solution are added. The mixture is diluted to the mark with solvent and shaken well. An appropriate amount is taken, the mixture is centrifuged, the supernatant is filtered, and the filtrate is obtained.

9. The method for detecting related substances in estradiol valerate tablets according to claim 8, characterized in that: The concentration of estradiol valerate in the mixed solution is 0.8-1.2 mg / ml.

10. The method for detecting related substances in estradiol valerate tablets according to claim 8, characterized in that: The content of impurities in the impurity reference stock solution is 0.8-1.2 mg / ml.

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