Method for analyzing related substances of tibolone tablets

Abstract

The invention discloses a method for analyzing related substances of tibolone tablets. The method comprises the following steps: detecting or separating a to-be-detected sample by adopting a high performance liquid chromatography; wherein the detection conditions of the liquid chromatography are as follows: octadecyl bonded silica gel is used as a filling agent of a chromatographic column; the mobile phase A is water; the mobile phase B is acetonitrile-methanol; in the mobile phase B, the volume ratio of acetonitrile to methanol is (75-85): (25-15); the elution mode of the liquid chromatography is linear gradient elution. The detection and separation method provided by the invention can effectively separate tibolone from impurities, can effectively detect a plurality of impurities of tibolone tablets, and is high in sensitivity, strong in specificity, low in cost and simple and safe to operate.

Description

Technical Field

[0001] The invention relates to the field of drug analysis and detection, and in particular to a method for analyzing related substances of tibolone tablets by utilizing high performance liquid chromatography. Background Art

[0002] Tibolone, chemical name: 7α-methyl-l7β-hydroxy-19-nor-17α-pregnane-5(10)-ene-20-yn-3-one, chemical structure:

[0003]

[0004] Tibolone is a synthetic steroid hormone drug used for hormone replacement therapy (HRT), mainly used to treat low estrogen symptoms caused by natural menopause or artificial menopause in women.

[0005] Tibolone tablets may produce degradation products during the process of production and storage. Therefore, establishing an effective analytical method for related substances in Tibolone tablets to quickly and accurately determine the related substances in Tibolone tablets has important practical significance in the quality control of the preparation production process.

[0006] The National Medical Products Administration (NMPA)'s import drug registration standard JX20120192 discloses three related substances of Tibolone Tablets, as shown in Table 1 below:

[0007] Table 1: Related substances disclosed in Tibolone tablets

[0008]

[0009] When reproducing the method of registration standards for imported drugs, it was found that:

[0010] (1) The detection sensitivity of the method is insufficient and is easily affected by baseline fluctuations;

[0011] (2) Using three impurity reference substances as external standards for quantification is costly;

[0012] (3) Its mobile phase uses tetrahydrofuran, which is easy to deteriorate and will slowly form explosives when in contact with air. It also has obvious corrosive and anesthetic effects. When inhaled by the human body, it will cause upper respiratory tract irritation and central nervous system depression. Long-term use is harmful to the safety and health of operators.

[0013] When reproducing the detection method for tibolone-related substances in the European Pharmacopoeia, it was found that the separation between impurities A and B was poor and the sensitivity of impurity C was not high.

[0014] Based on the above deficiencies, it is necessary to improve the analytical method of Tibolone Tablets-related substances and establish a set of sensitive, effective, affordable, safe and reliable analytical methods for Tibolone Tablets-related substances for quality control of intermediates and finished products. Summary of the invention

[0015] In view of the above-mentioned defects of the existing analysis methods, the technical problem solved by the present invention is to provide a method for analyzing related substances of tibolone tablets using high performance liquid chromatography, which can effectively separate tibolone from impurities (see Table 1 above for details), and can effectively detect multiple impurities in tibolone tablets, with high sensitivity, strong specificity, low cost, simple and safe operation.

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

[0017] The present invention provides a method for analyzing related substances of tibolone tablets, which comprises the following steps:

[0018] High performance liquid chromatography is used to detect or separate the sample to be tested; the sample to be tested includes tibolone and impurity 1, and the impurity 1 is selected from one or more of impurity A, impurity B and impurity C;

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

[0020] Chromatographic column: Octadecyl bonded silica gel as filler;

[0021] Mobile phase A was water;

[0022] Mobile phase B is an acetonitrile-methanol mixed solution; the volume ratio of acetonitrile to methanol is (75-85): (25-15);

[0023] The elution mode of the high performance liquid chromatography is linear gradient elution, and the elution procedure is as follows:

[0024] ;

[0025] wherein X1 is selected from 68-72%; X2 is selected from 38-42%; Y1 is selected from 30-35; Y2 is selected from 32-37; Y3 is selected from 40-44;

[0026] For example, in the table, "0→20 minutes, the volume of mobile phase A is X1→X2" means that in the range of 0-20 minutes, mobile phase A (the volume of mobile phase A is the percentage of the total volume of mobile phase) changes linearly from X1 to X2; "20→Y1 minutes, the volume of mobile phase B is 100-X2" means that in the range of 20-Y1 minutes, mobile phase B (the volume of mobile phase B is the percentage of the total volume of mobile phase) is a fixed value of 100-X2.

[0027] In one embodiment, the chromatographic column of the high performance liquid chromatography is Waters Nova-pak C18 or ChromCore120 C18.

[0028] In one embodiment, the filler particles of the HPLC column have a particle size of 2.5 µm to 7.5 µm, such as 5 or 4 µm.

[0029] In one embodiment, the length of the HPLC column is 100 mm to 250 mm, preferably 150 mm.

[0030] In one embodiment, the inner diameter of the HPLC column is 2 mm to 5 mm, for example, 4.6 or 3.9 mm.

[0031] In one embodiment, in the mobile phase B, acetonitrile-methanol = 80:20 (%, V / V).

[0032] In a certain embodiment, the elution procedure of the linear gradient elution is:

[0033] .

[0034] The flow rate of the HPLC method is the conventional flow rate of HPLC in the art, preferably 0.5 mL / min-1.5 mL / min, for example 1.0 mL / min.

[0035] The injection volume of the HPLC method is the conventional injection volume of HPLC in the art, preferably 5 μL to 20 μL, for example, 10 μL.

[0036] The column temperature of the HPLC method is a conventional column temperature of HPLC method in the art, preferably 35°C-40°C, such as 40°C.

[0037] The detector for the HPLC method is a detector conventionally used in the HPLC method in the art, preferably a DAD detector or a UV detector.

[0038] In one embodiment, the detection wavelength of the high performance liquid chromatography is 210 nm to 240 nm, preferably 240 nm.

[0039] In one embodiment, the detection time of the high performance liquid chromatography is 40-44 minutes, preferably 40 minutes.

[0040] The analysis method can be performed using a conventional liquid chromatograph in the art, preferably a Shimadzu or Thermo Fisher high performance liquid chromatograph.

[0041] In one embodiment, the high performance liquid chromatography method satisfies the following conditions:

[0042] Chromatographic column: Waters Nova-pak C18, 150*3.9mm, 4μm or ChromCore 120 C18, 4.6*150mm, 5μm;

[0043] Mobile phase A: water;

[0044] Mobile phase B: acetonitrile and methanol in a volume ratio of 80:20;

[0045] The elution mode of the high performance liquid chromatography is linear gradient elution, and the elution procedure is as follows:

[0046] ;

[0047] The following conditions are preferred:

[0048] Chromatographic column: Waters Nova-pak C18 (150*3.9mm, 4μm) or ChromCore 120 C18 (4.6*150mm, 5μm);

[0049] Column temperature: 40°C;

[0050] Mobile phase A: water;

[0051] Mobile phase B: acetonitrile-methanol = 80:20 (%, V / V);

[0052] Flow rate: 1.0 mL / min;

[0053] Injection volume: 10 μL;

[0054] Detection time: 40min;

[0055] Detector: DAD detector or UV detector;

[0056] Detection wavelength: 240nm;

[0057] The elution mode of the high performance liquid chromatography is linear gradient elution, and the elution procedure is as follows:

[0058] .

[0059] In the analytical method, the sample to be tested needs to undergo conventional pretreatment before elution to meet the injection standard, which may include the following steps: dissolving the sample to be tested in a solvent, and then filtering it with a polytetrafluoroethylene microporous filter membrane to obtain a filtrate; the solvent may be acetonitrile-water = 75:25 (%, V / V); the pore size of the filter membrane is preferably 0.2-0.22μm, for example 0.22μm.

[0060] The analysis method may further include using an external standard method to determine the content of each impurity, preferably, using impurity C as an external standard.

[0061] In the analytical method, when impurity C is used as an external standard and the content of each relevant impurity in the analyte is calculated by the external standard method, the calculation formula is as follows:

[0062]

[0063] Where:

[0064] M is the sample weight of the reference solution, mg;

[0065] P is the content of reference substance, %;

[0066] A 2 is the peak area of ​​the test solution;

[0067] V 2 is the dilution volume of the test sample, ml;

[0068] F is the correction factor of impurity A / B relative to impurity C (1.13 for impurity A, 1.23 for impurity B, and 1.00 for unknown impurity);

[0069] A 1 is the average peak area of ​​the reference solution;

[0070] V 1 is the dilution volume of the reference solution, ml;

[0071] S is the specification of the test product, mg / tablet;

[0072] N is the number of samples, pieces.

[0073] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0074] The reagents and raw materials used in the present invention are commercially available.

[0075] The positive and progressive effects of the present invention are:

[0076] The method described in the present invention is suitable for the separation and determination of impurities related to Tibolone tablets, and can be used to detect a certain substance alone, or to separate and detect Tibolone and its related impurities at the same time. Compared with the prior art, the analytical method of the present invention is safe, economical, and sensitive. It uses a single wavelength and an impurity C external standard to detect Tibolone tablet related substances (the three degradation impurities described in Table 1), and can well separate Tibolone from other inactive substances. The method has strong specificity, high sensitivity, good separation, and the tablet excipients do not interfere with the detection, which can better control the quality of Tibolone tablets and has significant progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is the test spectrum of the spiked solution of the test sample in Example 1 under the method of the present invention.

[0078] Figure 2 This is the test spectrum of the test solution in Comparative Example 1 under the imported registered standard method.

[0079] Figure 3 This is the test spectrum of the test solution of Comparative Example 2 under the British Pharmacopoeia method.

[0080] Figure 4 This is the detection spectrum of the test solution in Comparative Example 3.

[0081] Figure 5 This is the detection spectrum of the test solution of Comparative Example 4. DETAILED DESCRIPTION

[0082] 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.

[0083] The experimental instruments used in the following examples are all Shimadzu or Thermo Fisher high performance liquid chromatographs (UV detector / DAD detector). Unless otherwise specified, mobile phase A is pure water, mobile phase B is acetonitrile-methanol (volume ratio is 80:20), 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 (75:25); the injection volume is 10 μl; and the detection wavelength is 240 nm. The sources of the samples and reference substances involved in the examples are as follows:

[0084] Table 2: Sample and reference material information

[0085]

[0086] Example 1 Determination of Related Substances in Tibolone Tablets - Specificity

[0087] Chromatographic column: ChromCore 120 C18, 5 μm, 4.6*150 mm;

[0088] Detection wavelength: 240nm; flow rate: 1.0ml / min; column temperature: 40℃; injection volume: 10μl; solvent: acetonitrile-water (75:25);

[0089] Mobile phase: Mobile phase A is pure water, mobile phase B is acetonitrile-methanol (80:20), gradient elution:

[0090]

[0091] The elution gradient changes linearly. For example, in the above table, from 0 to 20 minutes, mobile phase A (the percentage of mobile phase A volume in the total volume of mobile phase) changes linearly from 70% to 40%, and at the same time, mobile phase B (the percentage of mobile phase B volume in the total volume of mobile phase) changes linearly from 30% to 60%, wherein the sum of mobile phase A and mobile phase B is always 100%. The rest of the embodiments are the same and will not be described in detail.

[0092] Tibolone blank excipient solution: Take 500 mg of tibolone blank excipient (without API), place it in a 25 ml volumetric flask, add an appropriate amount of solvent, ultrasonicate for 10 minutes, dilute to the scale with solvent, shake well, and then filter with a 0.22 μm polytetrafluoroethylene filter membrane, and take the filtrate as the blank excipient solution.

[0093] Impurity localization solutions: Take appropriate amounts of each impurity and dilute with solvent to prepare impurity A localization solution containing approximately 5 μg of impurity A, impurity B localization solution containing 5 μg of impurity B, and impurity C localization solution containing approximately 25 μg of impurity C per 1 ml.

[0094] Tibolone targeting solution: Take an appropriate amount of tibolone and dilute it with a solvent to make a targeting solution containing approximately 25 μg of tibolone per 1 ml.

[0095] Mixed localization solution: Weigh appropriate amounts of impurity A, impurity B, impurity C, and tibolone and place them in the same volumetric flask. Dilute with solvent to make a solution containing approximately 5 μg of impurity A and impurity B, 25 μg of impurity C and tibolone per 1 ml. Mix well to obtain a mixed localization solution.

[0096] Impurity mixed solution: Accurately weigh appropriate amounts of impurity A, impurity B, and impurity C and place them in the same volumetric flask. Dilute with solvent to make a solution containing approximately 25 μg of impurity A and impurity B and 125 μg of impurity C per 1 ml. Mix well to obtain the impurity mixed solution.

[0097] Test sample spiked solution: Take 5 Tibolone tablets (equivalent to approximately 12.5 mg Tibolone), place in a 25 ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, accurately measure 5 ml of the impurity mixed solution (impurity A: 25 μg / ml, impurity B: 25 μg / ml, impurity C: 125 μg / ml), dilute to the scale with solvent, shake well, filter with a 0.22 μm polytetrafluoroethylene filter membrane, and take the filtrate as the test sample spiked solution.

[0098] Accurately measure each solution and analyze it according to the above chromatographic conditions, and record the retention time, separation degree, etc. The specific results are as follows:

[0099]

[0100] Analysis of chromatographic results of test sample spiked solution:

[0101]

[0102] The results showed that under the above conditions, the response values ​​of impurities A / B / C in the spiked solution of the test sample were large at 240nm and the peak shape was good; the separation of each impurity was good and there was no interference from the air assist, indicating that the method had good specificity.

[0103] Example 2 Determination of Related Substances in Tibolone Tablets

[0104] Chromatographic column: Waters Nova-pak C18 150*3.9 mm, 4 μm; other chromatographic conditions are the same as in Example 1.

[0105] Test solution: Take 5 Tibolone tablets (equivalent to approximately 12.5 mg Tibolone), place in a 25 ml volumetric flask, add an appropriate amount of solvent, dissolve by ultrasonication, dilute to the scale with solvent, shake well, and filter with a 0.22 μm polytetrafluoroethylene filter membrane to obtain the solution.

[0106] Reference solution: Accurately weigh an appropriate amount of impurity C reference, dissolve it in solvent and quantitatively dilute it to make a reference solution containing approximately 25 μg of impurity C per 1 ml.

[0107] The above solution was sampled and analyzed, and the test results of the test solution are shown in the following table:

[0108]

[0109] Impurity C in the reference solution was injected five times, and its average peak area was 12.639.

[0110] The contents of tibolone and impurities in the test solution were calculated by the external standard method using impurity C as the reference substance and multiplying the correction factor. The calculation formula is as follows:

[0111]

[0112] M is the sample weight of the reference solution, mg;

[0113] P is the content of reference substance, %;

[0114] A 2 is the peak area of ​​the test solution;

[0115] V 2 is the dilution volume of the test sample, ml;

[0116] F is the correction factor of impurity A / B relative to impurity C (1.13 for impurity A, 1.23 for impurity B, and 1.00 for unknown impurity);

[0117] A 1 is the average peak area of ​​the reference solution;

[0118] V 1 is the dilution volume of the reference solution, ml;

[0119] S is the specification of the test product, mg / tablet;

[0120] N is the number of samples, pieces.

[0121] The calculated contents of each impurity are as follows:

[0122]

[0123] The peak shapes of each component were good, and the separation degree between tibolone and each impurity was good. The content of each impurity in tibolone tablets could be effectively detected by using impurity C external standard.

[0124] Example 3 Determination of related substances in Tibolone tablets - Destruction experiment

[0125] Chromatographic column: Waters Nova-pak C18 150*3.9mm, 4μm (other chromatographic conditions are the same as in Example 1)

[0126] Test solution: Take 5 Tibolone tablets (equivalent to approximately 12.5 mg Tibolone), place in a 25 ml volumetric flask, add solvent and ultrasonically dissolve them, destroy them to an appropriate degree under acid / alkali / high temperature conditions, dilute to the scale with solvent, and filter through a 0.22 μm polytetrafluoroethylene filter membrane to obtain the test solution.

[0127] The specific destruction conditions are as follows:

[0128] High temperature destruction: Take 5 Tibolone tablets (equivalent to approximately 12.5 mg Tibolone), place them in a 25 ml volumetric flask, add an appropriate amount of solvent, ultrasonicate for 10 minutes to dissolve, then place in an 80°C oven for 1 day, take out, dilute to the scale with solvent, and filter through a 0.22 μm polytetrafluoroethylene filter membrane.

[0129] Alkali destruction: Take 5 tibolone tablets (equivalent to approximately 12.5 mg of tibolone), place in a 25 ml volumetric flask, add 9 ml of solvent, sonicate for 10 minutes to dissolve, add 1 ml of 0.1 M NaOH, mix well, let stand for 30 minutes, neutralize with 0.1 M HCl, dilute to the scale with solvent, and filter with a 0.22 μm polytetrafluoroethylene filter membrane.

[0130] Acid destruction: Take 5 Tibolone tablets (equivalent to approximately 12.5 mg Tibolone), place in a 25 ml volumetric flask, add 7 ml of solvent, sonicate for 10 min to dissolve, add 3 ml of 0.1 M HCl, mix well, let stand for 3 hours, neutralize with 0.1 M NaOH to neutral, dilute to the scale with solvent, and filter with a 0.22 μm polytetrafluoroethylene filter membrane.

[0131] Reference solution: Take an appropriate amount of impurity C reference substance, weigh it accurately, dissolve it in solvent and quantitatively dilute it to make a reference solution containing approximately 25 μg of impurity C per 1 ml.

[0132] Take the above solution for sampling and analysis, calculate the impurity content and material conservation in the test solution under different destruction conditions, and the results are shown in the following table:

[0133]

[0134] (“-” indicates unknown impurities. Due to differences in the state of the instrument and chromatographic column, the destruction conditions may affect the properties of the test sample, such as pH, resulting in slightly different retention times under different tests. The impurities are located by the relative retention time (RRT) of each impurity relative to tibolone, where the RRT of impurity A is 0.35, the RRT of impurity B is 0.39, and the RRT of impurity C is 0.78.)

[0135] In the table below, the content of Tibolone after degradation was calculated by comparing it with the peak area of ​​the undestroyed sample.

[0136]

[0137] Impurities A, B, C and unknown impurities under various destruction conditions can be accurately quantified, and the degree of destruction and material conservation of impurity generation indicate that the detection capability of the method is sufficient.

[0138] Example 4 Determination of Related Substances in Tibolone Tablets - Accuracy Experiment

[0139] Test solution: Take 5 Tibolone tablets, place them in a 25 ml volumetric flask, add an appropriate amount of solvent, sonicate for 10 minutes to dissolve, add appropriate amounts of impurity mixed solution, dilute to the scale with solvent, shake well, filter with a 0.22 μm polytetrafluoroethylene filter membrane, and take the filtrate.

[0140] Under the proposed chromatographic conditions (same as in Example 1), the sample solution was sampled and analyzed, and the recovery rate was calculated three times using the ratio of the measured result to the theoretical value, and the average value was taken. The recovery rate of each impurity in the Tibolone tablets was calculated within the limit concentration range of 20% to 150%. Among them, the measured value was calculated using the external standard method, and the theoretical value was calculated based on the amount of impurities added to the sample solution. The specific results are shown in the following table.

[0141]

[0142] The results showed that the method met the recovery requirements (the recovery should be 92%~105%, RSD≤4.0%) and had good accuracy.

[0143] Example 5 Determination of Tibolone-Related Substances - Limit of Detection and Limit of Quantitation

[0144] Take impurities A, B and C of tibolone, dissolve them with solvents and dilute them step by step. Under the proposed chromatographic conditions (same as in Example 1), inject the diluted solutions for analysis, record the chromatograms, 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 the following table:

[0145]

[0146] The detection limit and quantification limit of each component were both below 0.05% of the test sample concentration (0.5 mg / ml). All impurities could be effectively detected and accurately quantified, and the method had good sensitivity.

[0147] Comparative Example 1: Determination of Related Substances in Tibolone Tablets - Import Registration Standard

[0148] Determination method: Determined according to the import registration standard JX20120192, the mobile phase is tetrahydrofuran-water (35:65); the flow rate is 1.0 ml / min; the chromatographic column is Waters Nova-pak C18, 150*3.9 mm 4μm; the column temperature is 40°C; the injection volume is 10μl; isocratic elution; the running time is 25 minutes; the detection wavelength is 240nm.

[0149] Test solution: Take 1 Tibolone tablet, grind it into powder and transfer it all to a centrifuge tube, add 5 ml of anhydrous ethanol, sonicate for 10 minutes, then centrifuge for 5 minutes, and take the supernatant.

[0150] Accurately measure 10 μl of the test solution and inject it into the liquid chromatograph to record the spectrum. The results are shown in the following table and the spectrum is attached. Figure 2 :

[0151]

[0152] The results showed that impurity A was close to the solvent peak and was easily affected by the baseline. The mobile phase used tetrahydrofuran, which was highly toxic and corrosive, and long-term use was harmful to the health of the experimenters. Anhydrous ethanol was easily volatile as a solvent and interfered with the quantification of the solution.

[0153] Comparative Example 2: Determination of Related Substances in Tibolone Tablets - BP

[0154] Determination method: According to the British Pharmacopoeia Tibolone detection method, the mobile phase is tetrahydrofuran-water (28:72); the flow rate is 1.0 ml / min; the chromatographic column is Waters Nova-pak C18, 150*3.9 mm 4μm; the column temperature is 40°C; the injection volume is 10μl; isocratic elution; the running time is 25 minutes; the detection wavelength is 240nm.

[0155] Test solution: Take 1 Tibolone tablet, grind it into powder and transfer it all to a centrifuge tube, add 5 ml of anhydrous ethanol, sonicate for 10 minutes, then centrifuge for 5 minutes, and take the supernatant.

[0156] Accurately measure 10 μl of the test solution and inject it into the liquid chromatograph to record the spectrum. The results are shown in the following table and the spectrum is attached. Figure 3 :

[0157]

[0158] Similar to the results of the imported registration standard, the mobile phase is tetrahydrofuran, which is harmful to the health of experimenters; the integration of impurity A is easily affected by baseline fluctuations; and the peak shape of impurity B is poor.

[0159] Comparative Example 3 Determination of Related Substances in Tibolone Tablets - Mobile Phase

[0160] Determination method: flow rate is 1.0ml / min; chromatographic column is Waters Nova-pak C18, 150*3.9mm, 4μm; column temperature is 40℃; injection volume is 10μl; running time is 10 minutes; detection wavelength is 240nm; mobile phase A is pure water, phase B is acetonitrile-methanol (40:10), phase A: phase B = 50:50, isocratic elution.

[0161] Test solution: Take 1 Tibolone tablet, grind it into powder and transfer it all to a centrifuge tube, add 5 ml of anhydrous ethanol, sonicate for 10 minutes, then centrifuge for 5 minutes, and take the supernatant.

[0162] Accurately measure 10 μl of the test solution and inject it into the liquid chromatograph to record the spectrum. The results are shown in the following table and the spectrum is attached. Figure 4 :

[0163]

[0164] The mobile phase was replaced with a safer mixture of water + acetonitrile + methanol, but the results showed that impurities A and B were not separated. At the same time, the baseline of impurity C at the peak was obviously drifting upward, and the integration was obviously disturbed.

[0165] Comparative Example 4 Determination of Related Substances in Tibolone Tablets - Gradient

[0166] Determination method: The chromatographic column is Waters Nova-pak C18, 150*3.9mm, 4μm; the detection wavelength is 240nm; the flow rate is 1.0mL / min; the column temperature is 40℃; the mobile phase A is pure water, the mobile phase B is acetonitrile-methanol (80:20), and the gradient elution is performed as shown in the following table:

[0167]

[0168] Test solution: Take 5 Tibolone tablets and put them into a 25 ml volumetric flask, add 75% acetonitrile, dissolve by ultrasonication, dilute to the scale with solvent, shake well, and filter with a 0.22 μm filter membrane.

[0169] Accurately measure 10 μl of the test solution and inject it into the liquid chromatograph to record the spectrum. The results are shown in the following table and the spectrum is attached. Figure 5 :

[0170]

[0171] The results showed that the peaks were well shaped and well separated. However, when the sample was tested repeatedly, impurities with less polarity were eluted in the next injection, affecting the impurity C integral.

Claims

1. A method for analyzing related substances in Tibolone tablets, characterized in that: It includes the following steps: High performance liquid chromatography is used to detect or separate the sample to be tested; the sample to be tested includes tibolone and impurity 1, and the impurity 1 is selected from one or more of impurity A, impurity B and impurity C; The detection conditions of the high performance liquid chromatography include: Chromatographic column: Octadecyl bonded silica gel as filler; Mobile phase A was water; Mobile phase B is an acetonitrile-methanol mixed solution; the volume ratio of acetonitrile to methanol is (75-85): (25-15); The elution mode of the liquid chromatography is linear gradient elution, and the elution procedure is as follows: ； Among them, X1 is selected from 68-72%; X2 is selected from 38-42%; Y1 is selected from 30-35; Y2 is selected from 32-37; and Y3 is selected from 40-44.

2. The analysis method according to claim 1, characterized in that The elution program of the linear gradient elution is: 。 3. The analysis method according to claim 1, characterized in that It meets one or more of the following conditions: (1) In the mobile phase B, the volume ratio of acetonitrile to methanol is 80:20; (2) The chromatographic column is Waters Nova-pak C18 or ChromCore 120 C18; (3) The filler particle size of the chromatographic column is 2.5~7.5μm; (4) The length of the chromatographic column is 100-250 mm; (5) The inner diameter of the chromatographic column is 2-5 mm; (6) The column temperature of the chromatographic column is 35-40°C; (7) The flow rate of the mobile phase is 0.5-1.5 mL / min; (8) The injection volume of the liquid chromatography method is 5~20μL.

4. The analysis 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 4 μm; (2) The length of the chromatographic column is 150 mm; (3) The inner diameter of the chromatographic column is 4.6 mm or 3.9 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 10 μL.

5. The analysis method according to claim 3, characterized in that The chromatographic column is Waters Nova-pak C18, 150*3.9mm, 4μm or ChromCore 120 C18, 4.6*150mm, 5μm.

6. The analysis method according to claim 1, characterized in that It meets one or more of the following conditions: (1) The detector of the high performance liquid chromatography is a DAD detector or a UV detector; (2) The detection wavelength of the high performance liquid chromatography is 210-240 nm, preferably 240 nm; (3) The detection time of the high performance liquid chromatography method is 40 to 44 minutes, preferably 40 minutes.

7. The analysis method according to claim 1, characterized in that The high performance liquid chromatography method meets the following conditions: Chromatographic column: Waters Nova-pak C18, 150*3.9mm, 4μm or ChromCore 120 C18, 4.6*150mm, 5μm; Mobile phase A: water; Mobile phase B: a mixed solution of acetonitrile and methanol, with a volume ratio of acetonitrile to methanol of 80:20; The elution mode of the high performance liquid chromatography is linear gradient elution, and the elution procedure is as follows: 。 8. The analysis method according to claim 1, characterized in that The high performance liquid chromatography method meets the following conditions: Chromatographic column: Waters Nova-pak C18, 150*3.9mm, 4μm or ChromCore 120 C18, 4.6*150mm, 5μm; Column temperature: 40°C; Mobile phase A: water; Mobile phase B: a mixed solution of acetonitrile and methanol, with a volume ratio of acetonitrile to methanol of 80:20; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Detection time: 40min; Detector: DAD detector or UV detector; Detection wavelength: 240nm; The elution mode of the high performance liquid chromatography is linear gradient elution, and the elution procedure is as follows: 。 9. The analysis method according to claim 1, characterized in that The sample to be tested needs to be pretreated before elution, which includes the following steps: dissolving the sample to be tested in a solvent, and then filtering it with a polytetrafluoroethylene microporous filter membrane to obtain a filtrate.

10. The analysis method according to claim 9, characterized in that It meets one or both of the following conditions: (1) The solvent is a mixed solution of acetonitrile and water, and the volume ratio of acetonitrile to water is preferably 75:25; (2) The pore size of the filter membrane is 0.2-0.22 μm, for example 0.22 μm.

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