An analytical method for dienogest

Abstract

The present invention discloses an analytical method for dienogest. The present invention provides an analytical method for dienogest, which comprises the following steps: adopting high performance liquid chromatography to perform gradient elution on the analyte to be measured, and that's it; the analyte to be measured contains dienogest. The analytical method provided by the present invention can effectively separate dienogest A impurity and K impurity, and the system pressure is moderate. It can effectively detect multiple impurities of dienogest, with high sensitivity and strong specificity, and can be further used to monitor the quality of dienogest final products or raw materials and preparations. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis and detection, and particularly to an analysis method for dienogest. Background Art

[0002] In pharmaceutical analysis, the so-called related substances refer to substances that are not the main components in a specific drug but are related to the main component. Since a drug goes through a relatively complex and long process from the synthesis of the active pharmaceutical ingredient to the preparation of the relevant preparations, and then through storage, transportation, and use, during each process, related substances may be generated. For example, starting materials, reagents, intermediates, by-products, and isomers may be introduced during production; degradation products, polymers, or crystal form transformations and other special impurities may be generated during storage and transportation. To ensure the safety and effectiveness of drugs and also consider the actual production situation, therefore, in the research of drugs at home and abroad, a certain limit of harmless or low-toxic related substances is allowed, but related substances with high toxicity, which can endanger human health, are ineffective, or can affect the stability of drugs must be strictly controlled. Therefore, the detection of related substances is an important indicator for controlling the quality of drugs.

[0003] Dienogest, chemical name: 17α-cyanomethyl-17β-hydroxy-13β-methylsteroid-4,9-diene-3-one, chemical structure:

[0004]

[0005] It is a mixed progestogen and has the pharmacological advantages of both natural and synthetic progestogens, and is mainly used for the treatment of endometriosis.

[0006] The existing high-performance liquid chromatography analysis method for related substances of dienogest tablets (import registration standard JX20170016) is very inconvenient to measure. Using this method, the system pressure is very high, and there are many problems such as Seco-dienogest not being detected, the resolution of impurity A and impurity K not being optimized, and the baseline being steep. Therefore, it is necessary to explore and improve the measurement conditions to establish a set of rapid, simple, effective, and reliable finished product reaction monitoring and related substance analysis methods.

[0007]

[0008] The present invention aims to at least solve one of the above technical problems to some extent or at least provide a useful commercial option. For this purpose, an object of the present invention is to propose a method for analyzing related substances of dienogest tablets by high-performance liquid chromatography, which can effectively separate impurity A and impurity K of dienogest, and the system pressure is moderate. Multiple impurities of dienogest can be effectively detected, with high sensitivity and strong specificity. Summary of the Invention

[0009] The technical problem to be solved by the present invention is that the existing analytical methods for dienogest cannot effectively analyze impurity K. To this end, the present invention provides an analytical method for dienogest. The analytical method provided by the present invention can effectively separate impurity A and impurity K of dienogest, and the system pressure is moderate. It can effectively detect multiple impurities of dienogest, with high sensitivity and strong specificity, and can be further used to monitor the quality of dienogest end products or raw materials and preparations.

[0010] The present invention provides an analytical method for dienogest, which includes the following steps: adopting high performance liquid chromatography to perform gradient elution on the analyte.

[0011] The stationary phase of the high performance liquid chromatography is octadecylsilyl bonded silica gel.

[0012] The mobile phase of the high performance liquid chromatography is mobile phase A and mobile phase B; mobile phase A is water and acetonitrile, and the volume ratio of water to acetonitrile is 95:5 - 85:15; mobile phase B is methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 70:30 - 80:20.

[0013] The analyte contains dienogest.

[0014] At the start of the gradient elution, the volume percentage of mobile phase A in the mobile phase is 65% - 75%; during the gradient elution, the volume percentage of mobile phase A in the mobile phase decreases by 0.8% - 1.2% per minute.

[0015] In the analytical method, the injection volume of the high performance liquid chromatography is the conventional injection volume in the art, preferably 1 - 100 μL, more preferably 40 - 80 μL, and most preferably 50 μL.

[0016] In the analytical method, the column temperature of the high performance liquid chromatography is the conventional column temperature in the art, preferably 35 - 45 °C, such as 45 °C or 40 °C.

[0017] In the analytical method, the detection wavelength of the high performance liquid chromatography is the conventional wavelength in the art, preferably 210 - 350 nm; more preferably 220 nm and / or 310 nm.

[0018] In the analytical method, the flow rate of the high performance liquid chromatography is the conventional flow rate in the art, preferably 0.8 - 1.2 ml / min, more preferably 1.0 ml / min.

[0019] In the described analysis method, the particle size of the octadecylsilyl-bonded silica gel can be 2.7 - 5 μm, such as 2.7 μm, 3.5 μm, or 5 μm.

[0020] In the described analysis method, the chromatographic column of the high performance liquid chromatography is a conventional chromatographic column in the art.

[0021] In the described analysis method, the length of the chromatographic column can be 100 - 200 mm, such as 150 mm.

[0022] In the described analysis method, the diameter of the chromatographic column can be 3 - 6 mm, such as 4.6 mm.

[0023] In the described analysis method, the model of the chromatographic column can be Agilent XDB-C18 (150*4.6 mm, 3.5 μm), Agilent XDB-C18 (150*4.6 mm, 5 μm), or WATERS X-Bridge C18 (150*4.6 mm, 3.5 μm).

[0024] In the described analysis method, in mobile phase A, the volume ratio of the water to the acetonitrile can be 90:10.

[0025] In the described analysis method, in mobile phase B, the volume ratio of the methanol to the acetonitrile can be 70:30, 75:25, or 80:20.

[0026] In the described analysis method, the analyte to be measured can be pretreated before elution to meet the injection standard. The pretreatment is a pretreatment under conventional conditions in the art. Preferably, the pretreatment includes the following steps: dissolving the analyte to be measured in a solvent.

[0027] In the pretreatment, the solvent is a conventional solvent in the art, preferably one or more of water, methanol, and acetonitrile, more preferably water and / or methanol, and most preferably water and methanol, wherein the volume ratio of methanol to water is 25:75.

[0028] In the pretreatment, the concentration of the analyte to be measured is a conventional concentration in the art, preferably 0.04 - 0.1 mg / mL, such as 0.05 mg / mL - 0.08 mg / mL.

[0029] In the described analysis method, the analyte to be measured may further contain substance A, and the substance A is one or more of Seco-Dino, A impurity, K impurity, G impurity, H impurity, the compound shown in Formula II, povidone K25, and C impurity;

[0030]

[0031] In the described analysis method, the analyte may further contain povidone. The povidone may be povidone K25.

[0032] In the described analysis method, the analyte may further contain substance B, and the substance B is K impurity and A impurity;

[0033]

[0034]

[0035] In the described analysis method, the analyte may further contain substance C, and the substance C is K impurity, A impurity and povidone;

[0036]

[0037] The povidone may be povidone K25.

[0038] In the described analysis method, the analyte may further contain substance D, and the substance D is K impurity and the compound shown in Formula II;

[0039]

[0040] In the described analysis method, the analyte may further contain substance E, and the substance E is K impurity, A impurity and the compound of Formula II;

[0041]

[0042]

[0043] In the described analysis method, the analyte may be dienogest tablets or dienogest bulk drug.

[0044] In the described analysis method, the dienogest tablets may contain povidone. The povidone may be povidone K25.

[0045] In the described analysis method, the dienogest tablets preferably have the following prescription:

[0046] Materials Prescription Quantity (mg) Dienogest 2 Lactose Monohydrate 62.8 Microcrystalline Cellulose 18 Potato Starch 36 Povidone K25 8.1 Magnesium Stearate 1.35 Talc Powder 4.05 Crospovidone 2.7 。

[0047] In the described analysis method, at the start of the gradient elution, the volume percentage of mobile phase A in the mobile phase may be 70%-75%.

[0048] In the described analysis method, during the gradient elution, the volume percentage of mobile phase A in the mobile phase may decrease by 1.0% per minute.

[0049] In the described analysis method, in the gradient elution, the volume percentage of the mobile phase A in the mobile phase can always decrease.

[0050] In the described analysis method, in the gradient elution, the volume percentage of the mobile phase A in the mobile phase can decrease linearly.

[0051] In the described analysis method, the time of the gradient elution can be determined according to the analysis target and analysis conditions, preferably 1 min to 18 min, more preferably 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min or 17 min.

[0052] In the described analysis method, the gradient elution can be any of the following:

[0053] Gradient elution 1

[0054]

[0055] Gradient elution 2

[0056]

[0057] Gradient elution 3

[0058]

[0059] Gradient elution 4

[0060]

[0061] Gradient elution 5

[0062]

[0063] Gradient elution 6

[0064]

[0065] In the described analysis method, the analyte to be measured may further contain substance A, and the substance A is one or more of Seco-dino, A impurity, K impurity, G impurity, H impurity, the compound shown in Formula II, povidone K25 and C impurity;

[0066]

[0067]

[0068] The gradient elution is as follows:

[0069]

[0070] In the said analysis method, the analyte to be measured may further contain Substance B, and the said Substance B is K impurity and A impurity;

[0071]

[0072] The said gradient elution is as described below:

[0073]

[0074] In the said analysis method, the analyte to be measured may further contain Substance C, and the said Substance C is K impurity, A impurity and povidone;

[0075]

[0076]

[0077] The said gradient elution is as described below:

[0078]

[0079] The said povidone may be K25.

[0080] In the said analysis method, the analyte to be measured may further contain Substance D, and the said Substance D is K impurity and the compound shown in Formula II;

[0081]

[0082] The said gradient elution is as described below:

[0083]

[0084] In the said analysis method, the analyte to be measured may further contain Substance E, and the said Substance E is K impurity, A impurity and the compound of Formula II;

[0085]

[0086] The said gradient elution is as described below:

[0087]

[0088] The said analysis method may be any of the following schemes:

[0089] Scheme 1:

[0090] The said mobile phase A is water and acetonitrile, and the volume ratio of the water and the acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of the methanol and the acetonitrile is 75:25; the column temperature of the high performance liquid chromatography is 40 °C; the injection volume of the high performance liquid chromatography is 50 μL;

[0091] The chromatographic column model is Agilent XDB-C18 (150*4.6 mm, 3.5 μm); the analyte to be measured is subjected to the following gradient elution:

[0092]

[0093] Option 2:

[0094] The mobile phase A is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 75:25; the column temperature of the high performance liquid chromatography is 40 °C; the injection volume of the high performance liquid chromatography is 50 μL;

[0095] The chromatographic column model is WATERS X-Bridge C18 (150*4.6 mm, 3.5 μm); the analyte to be measured is subjected to the following gradient elution:

[0096]

[0097] Option 3:

[0098] The mobile phase A is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 80:20; the column temperature of the high performance liquid chromatography is 45 °C; the injection volume of the high performance liquid chromatography is 50 μL;

[0099] The chromatographic column model is Agilent XDB-C18 (150*4.6 mm, 5 μm); the analyte to be measured is subjected to the following gradient elution:

[0100]

[0101] Option 4:

[0102] The mobile phase A is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 80:20; the column temperature of the high performance liquid chromatography is 40 °C; the injection volume of the high performance liquid chromatography is 50 μL;

[0103] The chromatographic column model is Agilent XDB-C18 (150*4.6 mm, 3.5 μm); the analyte to be measured is subjected to the following gradient elution:

[0104]

[0105] Option 5:

[0106] The mobile phase A is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 80:20; the column temperature of the high performance liquid chromatography is 40 °C; the injection volume of the high performance liquid chromatography is 10 μL;

[0107] The chromatographic column model is Agilent XDB-C18 (150*4.6 mm, 3.5 μm); the analyte to be measured is subjected to the following gradient elution:

[0108]

[0109] Scheme VI:

[0110] The mobile phase A is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 80:20; the column temperature of the high performance liquid chromatography is 40 °C; the injection volume of the high performance liquid chromatography is 50 μL;

[0111] The chromatographic column model is Agilent XDB-C18 (150*4.6 mm, 3.5 μm); the analyte to be measured is subjected to the following gradient elution:

[0112]

[0113] The analysis method can distinguish each component in substance F, and the substance F is dienogest, impurity K and impurity A;

[0114]

[0115] The analysis method can distinguish each component in substance G, and the substance G is dienogest, impurity K, impurity A and povidone, and the povidone can be K25;

[0116]

[0117] The analysis method can distinguish each component in substance H, and the substance H is dienogest, impurity K and the compound shown in formula II;

[0118]

[0119]

[0120] The analysis method can distinguish each component in substance I, and the substance I is dienogest, impurity K, impurity A and the compound of formula II;

[0121]

[0122] The analysis method can also distinguish the components in substance G, and the substance G is dienogest, Seco-dienogest, impurity A, impurity K, impurity G, impurity H, the compound shown in Formula II, povidone K25, and impurity C;

[0123]

[0124]

[0125] Among them, * represents a chiral center.

[0126] In the analysis method, the elution order of the components of the analyte can be Seco-dienogest → impurity A → impurity K → impurity G → impurity H → the compound shown in Formula II → impurity C.

[0127] On the basis of not violating the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain the preferred examples of the present invention.

[0128] The reagents and raw materials used in the present invention are all commercially available.

[0129] The positive and progressive effects of the present invention are as follows: The analysis method provided by the present invention can effectively separate dienogest from impurity A and impurity K, and the system pressure is moderate. It can effectively detect multiple impurities of dienogest, with high sensitivity and strong specificity, and can be further used to monitor the quality of dienogest end products, raw materials, and preparations. Detailed implementation manners

[0130] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the examples described herein. The experimental methods without specific conditions in the following examples are carried out according to the conventional conditions and methods, or selected according to the product specifications.

[0131] Dienogest tablets: Bayer (manufacturer), commercially available preparation

[0132] Prescription Type Prescription Quantity (mg) Proportion (%) Function Dienogest 2 1.5 Active Pharmaceutical Ingredient Lactose Monohydrate 62.8 46.5 Filler Microcrystalline Cellulose 18 13.3 Filler Potato Starch 36 26.7 Filler Povidone K25 8.1 6 Binder Magnesium Stearate 1.35 1 Lubricant and Glidant Talc Powder 4.05 3 Lubricant and Glidant Crospovidone 2.7 2 Disintegrant

[0133] The experimental instruments used in the following examples are all Shimadzu or Thermo Fisher high-performance liquid chromatographs (UV detectors). Unless otherwise specified, the chromatographic column used is Agilent XDB-C18, the column temperature is 40 °C; the injection volume is 50 μL; the detection wavelengths are 220 nm and 310 nm, the flow rate is 1.0 ml / min; the solvent for diluting the sample is methanol-water (25:75).

[0134] Example 1 Determination of related substances in dienogest tablets

[0135] Chromatographic conditions:

[0136] Mobile phase: Mobile phase A is water - acetonitrile (90:10), mobile phase B is methanol - acetonitrile (75:25), gradient elution; Chromatographic column: Agilent Eclipse XDB - C18 (150*4.6 mm, 3.5 μm); The elution gradient is as follows:

[0137]

[0138] Among them, the elution gradient changes linearly. For example, from 0 to 25 minutes in the above table, mobile phase A (the percentage of the volume of mobile phase A in the total volume of the mobile phase) changes linearly from 70% to 45%, and at the same time, mobile phase B (the percentage of the volume of mobile phase B in the total volume of the mobile phase) changes linearly from 30% to 55%, where the sum of mobile phase A and mobile phase B is always 100%. The same applies to the rest of the examples and will not be explained further.

[0139] Impurity mixed solution: Weigh appropriate amounts of Seco - dienogest, Compound II, Impurity A, Impurity K, Impurity G, Impurity H, Compound II, Impurity C and dienogest tablet powder respectively, dilute with a solvent and make a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0140]

[0141] Precisely measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0142] After comparison with the reference substance, the results of each peak are shown in the following table:

[0143] Serial Number Component Retention Time (min) Resolution with Adjacent Peak (220nm) Resolution with Adjacent Peak (310nm) 1 Seco-Dienogest 3.252 - - 2 Impurity A 5.270 11.20 - 3 Impurity K 5.822 2.69 2.70 4 Dienogest 12.995 2.12 2.17 5 Impurity G 13.923 3.06 - 6 Impurity H 14.938 3.32 - 7 Compound of Formula II 16.213 2.15 - 8 Impurity C 16.980 2.88 -

[0144] From the results in the above table, it can be seen that this analytical method can effectively detect Seco - dienogest, Compound II, Impurity A and Impurity K under the detection condition of 220 nm; under the detection condition of 310 nm, it can effectively detect Impurity A and Impurity K.

[0145] Example 2 Determination of related substances in dienogest tablets

[0146] Chromatographic conditions:

[0147] Mobile phase: Mobile phase A is water - acetonitrile (90:10), mobile phase B is methanol - acetonitrile (75:25)

[0148] Chromatographic column: WATERS X - Bridge C18 (150*4.6 mm, 3.5 μm); The elution gradient is as follows:

[0149]

[0150] Impurity mixed solution: Weigh appropriate amounts of Seco-dienogest, impurity A, impurity K, impurity G, impurity H, Compound II, impurity C and dienogest tablet powder respectively, dilute with a solvent and prepare a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0151] Precisely measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0152] After comparison with the reference standards, the results of each peak are shown in the following table:

[0153] Serial Number Component Retention Time (min) Resolution with Adjacent Peak (220nm) Resolution with Adjacent Peak (310nm) 1 Seco-Dienogest 3.352 - - 2 Impurity A 5.337 10.23 - 3 Impurity K 5.747 1.90 1.92 4 Dienogest 11.643 1.70 1.60 5 Impurity G 12.560 3.10 - 6 Impurity H 13.393 2.79 - 7 Compound of Formula II 14.385 2.25 - 8 Impurity C 15.057 2.48 -

[0154] From the results in the above table, it can be seen that under the detection conditions of 220 nm, this analytical method can effectively detect Seco-dienogest, Compound II, impurity A and impurity K; under the detection conditions of 310 nm, it can effectively detect impurity A and impurity K.

[0155] Example 3 Determination of related substances of dienogest tablets

[0156] Chromatographic conditions:

[0157] Mobile phase: Mobile phase A is water-acetonitrile (90:10), mobile phase B is methanol-acetonitrile (80:20), and the elution gradient is as follows:

[0158]

[0159] Chromatographic column: Agilent Eclipse XDB-C18 (150 * 4.6 mm, 5 μm);

[0160] Column temperature: 45 °C;

[0161] Impurity mixed solution: Weigh appropriate amounts of Seco-dienogest, impurity A, impurity K, impurity G, impurity H, Compound II, impurity C and dienogest tablet powder respectively, dilute with a solvent and prepare a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0162] Precisely measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0163] After comparison with the reference standards, the results of each peak are shown in the following table:

[0164] Serial Number Component Retention Time (min) Resolution with Adjacent Peak (220nm) Resolution with Adjacent Peak (310nm) 1 Seco-Dienogest 2.051 - - 2 Impurity A 4.085 13.52 - 3 Impurity K 4.487 1.54 1.50 4 Dienogest 8.867 2.22 2.07 5 Impurity G 9.460 3.34 - 6 Impurity H 10.107 3.86 - 7 Compound of Formula II 10.624 1.58 - 8 Impurity C 11.133 1.46 -

[0165] As can be seen from the results in the above table, under the detection condition of 220 nm, this analysis method can effectively detect Seco-dienogest, Compound II, Impurity A and Impurity K; under the detection condition of 310 nm, it can effectively detect Impurity A and Impurity K.

[0166] Example 4: Determination of Related Substances in Dienogest Tablets

[0167] Chromatographic conditions:

[0168] Mobile phase: Mobile phase A is water - acetonitrile (90:10), mobile phase B is methanol - acetonitrile (70:30), and the elution gradient is:

[0169]

[0170]

[0171] Chromatographic column: Agilent Eclipse XDB-C18 (150 * 4.6 mm, 3.5 μm);

[0172] Impurity mixed solution: Weigh appropriate amounts of Seco-dienogest, Impurity A, Impurity K, Impurity G, Impurity H, Compound II, Impurity C and dienogest tablet powder respectively, dilute with a solvent and prepare a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0173] Precisely measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0174] After comparison with the reference standards, the results of each peak are shown in the following table:

[0175] Serial Number Component Retention Time (min) Resolution with Adjacent Peak (220nm) Resolution with Adjacent Peak (310nm) 1 Seco-Dienogest 2.152 - - 2 Impurity A 3.980 12.32 - 3 Impurity K 4.350 1.83 1.77 4 Dienogest 9.630 2.11 2.12 5 Impurity G 10.610 4.72 - 6 Impurity H 11.343 3.69 - 7 Compound of Formula II 11.864 1.91 - 8 Impurity C 12.417 2.15 -

[0176] As can be seen from the results in the above table, under the detection condition of 220 nm, this analysis method can effectively detect Seco-dienogest, Compound II, Impurity A and Impurity K; under the detection condition of 310 nm, it can effectively detect Impurity A and Impurity K.

[0177] Example 5

[0178] Mobile phase: Mobile phase A is water - acetonitrile (90:10), mobile phase B is methanol - acetonitrile (75:25), and the elution gradient is:

[0179]

[0180] Chromatographic column: Agilent Eclipse XDB-C18 (150 * 4.6 mm, 3.5 μm)

[0181] Impurity mixed solution: Weigh appropriate amounts of Seco-dienogest, Impurity A, Impurity K, Impurity G, Impurity H, Compound II, Impurity C and dienogest tablet powder respectively, dilute with a solvent and prepare a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0182] Precisely measure 10 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0183] After comparison with the reference standards, the results of each peak are shown in the following table:

[0184] Serial Number Component Retention Time (min) Resolution with Adjacent Peak (220nm) Resolution with Adjacent Peak (310nm) 1 Seco-Dienogest Not Detected - - 2 Impurity A 5.264 - - 3 Impurity K 5.820 2.73 2.81 4 Dienogest 12.980 2.10 2.20 5 Impurity G 13.923 3.12 - 6 Impurity H 14.935 3.44 - 7 Compound of Formula II 16.217 2.16 - 8 Impurity C 16.975 2.72 -

[0185] It can be seen from the results in the above table that under the detection condition of 220 nm, this analytical method can effectively detect Compound II, Impurity A and Impurity K, but Seco-dienogest is not detected (below LOQ), and the detection sensitivity is slightly poor; under the detection condition of 310 nm, Impurity A and Impurity K can be effectively detected.

[0186] Example 6

[0187] Mobile phase: Mobile phase A is water - acetonitrile (90:10), mobile phase B is methanol - acetonitrile (75:25), gradient elution;

[0188] Gradient program:

[0189]

[0190] Chromatographic column: Agilent Eclipse XDB-C18 (150 * 4.6 mm, 3.5 μm)

[0191] Test solution: Weigh appropriate amounts of Seco-dienogest, Impurity A, Impurity K, Impurity G, Impurity H, Compound II, Impurity C and dienogest tablet powder respectively, dilute with a solvent and prepare a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml, mix well and filter to obtain the test solution.

[0192] Measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0193] After comparison with the reference standards, the results of each peak are shown in the following table:

[0194]

[0195] It can be seen from the results in the above table that under the detection condition of 220 nm, this analytical method can effectively detect Compound II, Impurity A and Impurity K, but Seco-dienogest can be detected, but there is interference from the solvent peak and accurate quantification cannot be carried out; under the detection condition of 310 nm, Impurity A and Impurity K can be effectively detected.

[0196] Determination of Related Substances in Dienogest Tablets of Comparative Example 1

[0197] Test method: Determined according to the method of the imported registration standard JX20170016. Mobile phase: Mobile phase A is water, and mobile phase B is methanol; flow rate is 0.8 ml / min; gradient program:

[0198]

[0199] Chromatographic column: Ascientis Express C18 (100 * 3.0 mm, 2.7 μm)

[0200] Impurity mixed solution: Weigh appropriate amounts of Seco-dienogest, Impurity A, Impurity K, Impurity G, Impurity H, Compound II, Impurity C and dienogest tablet powder respectively, dilute with solvent and prepare a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml, mix well and filter to obtain the impurity mixed solution.

[0201] Precisely measure 50 μl of the impurity mixed solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0202] After comparison with the reference standard, the results of each peak are shown in the following table:

[0203]

[0204]

[0205] The results show that Seco-dienogest was not detected, the separation of Impurity A and K, Impurity H, Compound II and Impurity C was not preferred, the system pressure was very high (greater than 40 MPa), and the baseline was relatively steep.

[0206] Comparative Example 2

[0207] Test method: Determined according to the method in the European Pharmacopoeia (EUROPEAN PHARMACOPOEIA 10.0; pages 2400 - 2402). Mobile phase: Mobile phase A is water - acetonitrile (90:10), and mobile phase B is water - acetonitrile (10:90); column temperature is 35 °C; detection wavelength is 210 nm; injection volume is 15 μl; gradient program:

[0208]

[0209] Chromatographic column: Agilent Eclipse XDB - C18 (150 * 4.6 mm, 3.5 μm);

[0210] Test solution: Weigh appropriate amounts of Seco-dienogest, impurity A, impurity K, impurity G, impurity H, compound of formula II, impurity C and dienogest tablets powder respectively, dilute with a solvent and prepare a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml. Mix well and filter to obtain the test solution.

[0211] Precisely measure 15 μl of the mixed impurity solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0212] After comparison with the reference standard, the results of each peak are shown in the following table:

[0213]

[0214]

[0215] The results show that under this method, the excipients of dienogest tablets overlap with the signals of each impurity, and effective detection cannot be achieved. Among them, the excipient povidone K25 has a response at 210 - 220 nm, and its interference with impurity detection is relatively large.

[0216] Comparative Example 3

[0217] Mobile phase: Mobile phase A is water, mobile phase B is methanol - acetonitrile (90:10), gradient elution;

[0218] Gradient program:

[0219]

[0220] Chromatographic column: Ascientis Express C18 (100 * 3.0 mm, 2.7 μm)

[0221] Test solution: Weigh appropriate amounts of Seco-dienogest, impurity A, impurity K, impurity G, impurity H, compound of formula II, impurity C and dienogest tablets powder respectively, dilute with a solvent and prepare a solution containing about 0.24 μg of each impurity and 80 μg of dienogest per 1 ml. Mix well and filter to obtain the test solution.

[0222] Measure 50 μl of the mixed impurity solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0223] After comparison with the reference standard, the results of each peak are shown in the following table:

[0224]

[0225]

[0226] The results show that there is interference in the detection of Seco-dienogest under this method, and effective separation of impurity A, impurity K and impurity H, compound of formula II cannot be achieved. In addition, the column pressure of the chromatographic column is very high.

[0227] Comparative Example 4

[0228] Mobile phase: Mobile phase A is water, and mobile phase B is methanol - acetonitrile (80:20). The column temperature is 45°C, and gradient elution is used; Gradient program:

[0229]

[0230] Chromatographic column: Ascientis Express C18 (100 * 3.0 mm, 2.7 μm)

[0231] Test solution: Weigh appropriate amounts of Seco - dienol, impurity A, impurity K, impurity G, impurity H, Compound II, impurity C, and dienogest tablet powder respectively, dilute with a solvent and prepare a solution containing approximately 0.24 μg of each impurity and 80 μg of dienogest per 1 ml. Mix well and filter to obtain the test solution.

[0232] Measure 50 μl of the mixed impurity solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0233] After comparison with the reference standard, the results of each peak are shown in the following table:

[0234]

[0235]

[0236] The results show that there is interference in the detection of Seco - dienol under this method, and the resolution between impurity A, impurity K, impurity H, and Compound II does not reach 1.5; in addition, the column pressure of the chromatographic column is very high.

[0237] Comparative Example 5

[0238] Mobile phase: Mobile phase A is water - acetonitrile (90:10), and mobile phase B is methanol - acetonitrile (75:25); Phase A: Phase B (55:45) isocratic elution for 60 min;

[0239] Chromatographic column: Agilent Eclipse XDB - C18 (150 * 4.6 mm, 5 μm),

[0240] Test solution: Weigh appropriate amounts of Seco - dienol, impurity A, impurity K, impurity G, impurity H, Compound II, impurity C, and dienogest tablet powder respectively, dilute with a solvent and prepare a solution containing approximately 0.24 μg of each impurity and 80 μg of dienogest per 1 ml. Mix well and filter to obtain the test solution.

[0241] Measure 50 μl of the mixed impurity solution and inject it into the liquid chromatograph to record the retention time, resolution, etc.

[0242] After comparison with the reference standard, the results of each peak are shown in the following table:

[0243] Serial Number Component Retention Time (min) S / N Number of Theoretical Plates 1 Seco-Dienogest 2.172 7.2 1455 2 Impurity A 4.343 10.9 2370 3 Impurity K 5.145 9.5 2217 4 Dienogest 10.565 - 8875 5 Impurity G 11.655 17.8 4533 6 Impurity H 12.787 8.9 4650 7 Compound of Formula II 12.980 13.2 5033 8 Impurity C 13.995 11.4 4988

[0244] The results show that the main problem with this method is that the peak shapes of the impurity peaks are poor, the number of theoretical plates is very low, the sensitivity is insufficient, it is difficult to achieve accurate quantification of trace impurities, and it cannot be used for the quality control of dienogest tablets.

Claims

1. An analytical method for dienogest related substances, characterized in that, It includes the following steps: Using high performance liquid chromatography, gradient elution of the analyte can be carried out. The stationary phase of the high performance liquid chromatography is octadecylsilyl bonded silica gel. The mobile phase of the high performance liquid chromatography is mobile phase A and mobile phase B; mobile phase A is water and acetonitrile, and the volume ratio of water to acetonitrile is 90:10; mobile phase B is methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 70:30 or 80:

20. The analyte is dienogest tablets or dienogest bulk drug. The gradient program is: In the analysis method, the analyte also includes Seco-dienogest, A impurity, K impurity, G impurity, H impurity, the compound shown in Formula II, povidone K25, and C impurity.

2. The analysis method according to claim 1, characterized in that, It meets one or more of the following conditions: a) In the analysis method, the injection volume of the high performance liquid chromatography is 1 - 100 μL. b) In the analysis method, the column temperature of the high performance liquid chromatography is 35 - 45 °C. c) In the analysis method, the detection wavelength of the high performance liquid chromatography is 210 - 350 nm. d) In the analysis method, the flow rate of the high performance liquid chromatography is 0.8 - 1.2 ml / min. e) In the analysis method, the particle size of the octadecylsilyl bonded silica gel is 2.7 - 5 μm. f) In the analysis method, the length of the chromatographic column of the high performance liquid chromatography is 100 - 200 mm. g) In the analysis method, the diameter of the chromatographic column of the high performance liquid chromatography is 3 - 6 mm. h) In the analysis method, the analyte is pretreated before elution, and the pretreatment includes the following steps: dissolving the analyte in a solvent.

3. The analysis method according to claim 2, characterized in that It meets one or more of the following conditions: i) In the analysis method, the injection volume of the high performance liquid chromatography is 40 - 80 μL. j) In the analysis method, the column temperature of the high performance liquid chromatography is 45 °C or 40 °C. k) In the analysis method, the detection wavelength of the high performance liquid chromatography is 220 nm and / or 310 nm. l) In the analysis method, the flow rate of the high performance liquid chromatography is 1.0 ml / min. m) In the analysis method, the particle size of the octadecylsilyl bonded silica gel is 2.7 μm, 3.5 μm or 5 μm. n) In the analysis method, the length of the chromatographic column of the high performance liquid chromatography is 150 mm. o) In the analysis method, the diameter of the chromatographic column of the high performance liquid chromatography is 4.6 mm. p) In the analysis method, the analyte is pretreated before elution, and the pretreatment includes the following steps: dissolving the analyte in a solvent, and the solvent is one or more of water, methanol, and acetonitrile. q) In the pretreatment, the concentration of the analyte is 0.04 - 0.1 mg / mL.

4. The analysis method according to claim 3, characterized in that, It meets one or more of the following conditions: r) In the analysis method, the injection volume of the high performance liquid chromatography is 50 μL. s) In the described analysis method, the model of the chromatographic column is Agilent XDB-C18, with a specification of 150*4.6 mm, 3.5 μm, Agilent XDB-C18, with a specification of 150*4.6 mm, 5 μm, or WATERS X-Bridge C18, with a specification of 150*4.6 mm, 3.5 μm; t) In the described analysis method, the analyte to be measured is pretreated before elution. The pretreatment includes the following steps: dissolving the analyte to be measured in a solvent, and the solvent is water and methanol, where the volume ratio of methanol to water is 25:75; In the pretreatment, the concentration of the analyte to be measured is 0.05 mg / mL - 0.08 mg / mL.

5. The analysis method according to claim 1, characterized in that The dienogest tablets contain povidone, and the povidone is povidone K25.

6. The analysis method according to claim 1, characterized in that The prescription of the dienogest tablets is as follows: 。 7. The analysis method according to claim 1, wherein It is any of the following schemes: Scheme 1: The mobile phase A is water and acetonitrile, and the volume ratio of water to acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 80:20; the column temperature of the high performance liquid chromatography is 45 °C; the injection volume of the high performance liquid chromatography is 50 μL; Scheme 2: The mobile phase A is water and acetonitrile, and the volume ratio of water to acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 80:20; the column temperature of the high performance liquid chromatography is 40 °C; the injection volume of the high performance liquid chromatography is 50 μL; Scheme 3: The mobile phase A is water and acetonitrile, and the volume ratio of water to acetonitrile is 90:10; the mobile phase B is methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 80:20; the column temperature of the high performance liquid chromatography is 40 °C; the injection volume of the high performance liquid chromatography is 10 μL.

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