Method for determining related substances of dehydropregnenolone acetate by utilizing HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for determining related substances of dehydropregnenolone acetate by utilizing HPLC (High Performance Liquid Chromatography), and belongs to the technical field of medical analysis. The method comprises the following specific steps: 1) preparing a reference solution and a test solution; 2) setting high performance liquid detection conditions; a chromatographic column taking octadecyl bonded silica gel as a filling agent is adopted; the mobile phase is a mixed solution of a mobile phase A and a mobile phase B, the mobile phase is used for isocratic elution, the mobile phase A is water, and the mobile phase B is acetonitrile; and (3) respectively sucking the test solution and the reference solution, injecting the test solution and the reference solution into a liquid chromatograph, recording and observing a chromatogram, and calculating the related substances in the dehydropregnenolone acetate. According to the present invention, with the method, the related substances in the dehydropregnenolone acetate can be rapidly, effectively, accurately and reliably separated and detected, and the quality of the dehydropregnenolone acetate can be controlled so as to control the quality of the epristeride synthesized by using the dehydropregnenolone acetate as the raw material, such that the medication safety of the patient can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis, and in particular relates to a method for determining related substances of dienol ketone acetate by utilizing HPLC. Background Art

[0002] 16-pregnadien-20-one acetate is chemically named 3β-hydroxy-pregnadien-5,16-diene-20-one-3-acetate, and its molecular formula is C 23 H 32 O 3 . Dienolone acetate is a structural intermediate of steroid hormones and can be used to synthesize a variety of drugs such as adrenal hormones, steroidal anti-inflammatory drugs, glucocorticoids, sex hormones, etc. Domestic manufacturers generally use diosgenin or radix sapogenins as raw materials to synthesize dienolone acetate through ring-opening acylation and oxidative hydrolysis. In the production of dienolone acetate, due to the repeated use of the mother liquor and the effect of excessive oxidation, the content of dienolone acetate has been around 98%. Dienolone acetate is the starting material for the synthesis of Aprelet. Aprelet is a new type of non-competitive 5α-reductase inhibitor that can specifically or selectively inhibit type II enzymes, but has a weaker effect on type I enzymes. Therefore, the drug mainly works in the prostate and can effectively reduce the content of dihydrotestosterone. Therefore, the quality control of dienolone acetate plays a key role in the quality of Aprelet, and the control of related substances is the key to quality control.

[0003] Therefore, it is necessary to provide a method for determining the related substances of dienol ketone acetate using HPLC. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for determining the related substances of dienol ketone acetate by using HPLC, which can quickly, effectively, accurately and reliably separate and detect the related substances in dienol ketone acetate, control the quality of dienol ketone acetate, thereby controlling the quality of Aprilate synthesized with dienol ketone acetate as raw material, and improving the safety of patients' medication.

[0005] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0006] A method for determining related substances of dienolone acetate by HPLC, the specific steps are as follows:

[0007] 1) Prepare reference solution and test solution;

[0008] 2) Setting high performance liquid chromatography detection conditions; using a chromatographic column with octadecyl bonded silica as a filler; using a mobile phase as a mixed solution of mobile phase A and mobile phase B, and performing isocratic elution with the mobile phase, wherein mobile phase A is water and mobile phase B is acetonitrile;

[0009] 3) Pipette the test solution and reference solution separately, inject them into the liquid chromatograph, record and observe the chromatogram, and calculate the related substances in dienol ketone acetate.

[0010] Further, in step 1), the preparation method of the test solution is: accurately weigh the dienol ketone acetate and place it in a volumetric flask, add methanol to dilute and fix the volume to a solution containing 1 mg of dienol ketone acetate per 1 mL; the preparation method of the reference solution is: accurately weigh the test solution and place it in a volumetric flask, add methanol to dilute and fix the volume to a solution containing 10 μg of dienol ketone acetate per 1 mL.

[0011] Furthermore, in step 2), the chromatographic column is GL InertSustain C18, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm.

[0012] Furthermore, in step 2), the volume ratio of water to acetonitrile is 15:85.

[0013] Furthermore, in step 2), the column temperature of the chromatographic column is 35°C.

[0014] Furthermore, in step 2), the flow rate of the mobile phase during the detection operation is 1 mL / min.

[0015] Furthermore, in step 2), the flow rate of isocratic elution is 1 mL / min.

[0016] Furthermore, in step 2), the isocratic elution time is 45 min.

[0017] Furthermore, in step 3), the injection volume of the test solution and the reference solution is 20 μL.

[0018] Furthermore, in step 3), the detection wavelengths of the liquid chromatograph are 207 nm and 240 nm.

[0019] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0020] (1) The present invention can quickly, effectively, accurately and reliably separate and detect related substances in dienol ketone acetate, control the quality of dienol ketone acetate, and thus control the quality of aplidite synthesized with dienol ketone acetate as a raw material, thereby improving the safety of medication for patients.

[0021] (2) The test method of the present invention has sufficient separation between dienol ketone acetate and impurities, does not interfere with the determination of dienol ketone acetate and impurities, and has excellent accuracy, injection precision, intermediate precision and durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the standard curve of dienol ketone acetate (207nm);

[0023] Figure 2 is the standard curve of dienolone acetate (240nm);

[0024] Figure 3 This is the standard curve of SM1-1;

[0025] Figure 4 This is the standard curve of SM1-2;

[0026] Figure 5 The system suitability spectrum is 207nm;

[0027] Figure 6 The system suitability spectrum is 240nm;

[0028] Figure 7 It is the undestructed-destructive test spectrum at 207nm;

[0029] Figure 8 It is the undestructed-destructive test spectrum at 240nm;

[0030] Fig. 9 It is the oxidation damage-destructive test spectrum at 207nm;

[0031] Fig.10 It is the oxidation damage-destructive test spectrum at 240nm;

[0032] Fig.11 It is the high temperature damage-destructive test spectrum at 207nm;

[0033] Fig.12 It is the high temperature damage-destructive test spectrum at 240nm;

[0034] Fig.13 It is the pyrolysis damage-destructive test spectrum at 207nm;

[0035] Fig.14 It is the pyrolysis damage-destructive test spectrum at 240nm;

[0036] Fig.15 It is the 207nm light damage-destructive test spectrum;

[0037] Fig.16It is the 240nm light damage-destructive test spectrum;

[0038] Fig.17 It is the acid damage-destructive test spectrum at 207nm;

[0039] Fig.18 It is the acid damage-destructive test spectrum at 240nm;

[0040] Fig.19 It is the alkaline damage-destructive test spectrum at 207nm;

[0041] Fig. 20 It is the 240nm alkaline damage-destructive test spectrum;

[0042] Fig.21 It is 207nm-different flow rate 1-durability spectrum;

[0043] Fig. 22 It is 240nm-different flow rate 1-durability spectrum;

[0044] Fig.23 It is 207nm-different flow rate 2-durability spectrum;

[0045] Fig.24 It is 240nm-different flow rate 2-durability spectrum;

[0046] Fig.25 It is 207nm-different column temperature 1-durability spectrum;

[0047] Fig.26 It is 240nm-different column temperature 1-durability spectrum;

[0048] Fig. 27 It is 207nm-different column temperature 2-durability spectrum;

[0049] Fig.28 It is 240nm-different column temperature 2-durability spectrum;

[0050] Fig.29 It is 207nm-different ratio 1-durability spectrum;

[0051] Fig.30 It is 240nm-different ratio 1-durability spectrum;

[0052] Fig.31 It is 207nm-different ratio 2-durability spectrum;

[0053] Fig.32 It is 240nm-different ratio 2-durability map;

[0054] Fig.33It is 207nm-different chromatographic columns-durability spectrum;

[0055] Fig.34 This is a 240nm-different chromatographic column-durability spectrum. DETAILED DESCRIPTION

[0056] The present invention is further described below in conjunction with specific embodiments.

[0057] The HPLC detection conditions used in the following examples are as follows:

[0058] Chromatographic column: GL InertSustain C18, 4.6 mm × 250 mm, 5 μm.

[0059] Flow rate: 1 mL / min;

[0060] Column temperature: 35°C;

[0061] Mobile phase: water-acetonitrile (volume ratio 15:85);

[0062] Detection wavelengths: 207nm and 240nm.

[0063] Injection volume: 20 μL

[0064] The mobile phase was used for isocratic elution, the flow rate of the isocratic elution was 1 mL / min, and the time of the isocratic elution was 45 min.

[0065] The related substance analysis method of the dienol ketone acetate of the present invention is verified by referring to the general rules of the four volumes of the 2015 edition of the Chinese Pharmacopoeia, relevant technical guidelines, etc.

[0066] The structural formula of SM described in the following examples is:

[0067]

[0068] The structural formula of SM1-1 is:

[0069]

[0070] The structural formula of SM1-2 is:

[0071]

[0072] The limit requirements calculated by the self-control method of the present invention are: the content of SM1-1 and SM1-2 shall not exceed 1.0%, the maximum unknown single impurity shall not exceed 1.5%, and the total impurity shall not exceed 2.0%.

[0073] Example 1

[0074] A method for determining related substances of dienolone acetate using HPLC:

[0075] (1) Prepare reference solution and test solution. The test solution is prepared by accurately weighing dihydrogen ketone acetate into a volumetric flask, adding methanol to dilute to a volumetric volume of 1 mg dihydrogen ketone acetate per 1 mL. The reference solution is prepared by accurately weighing the test solution into a volumetric flask, adding methanol to dilute to a volumetric volume of 10 μg dihydrogen ketone acetate per 1 mL.

[0076] (2) Setting the HPLC detection conditions; using octadecyl bonded silica gel as a chromatographic column, the chromatographic column is GL InertSustain C18, the chromatographic column length is 250 mm, the inner diameter is 4.6 mm, and the filler particle size is 5 μm; the column temperature of the chromatographic column is 35°C. The mobile phase is a mixed solution of mobile phase A and mobile phase B, and the mobile phase is used for isocratic elution, and the flow rate of the isocratic elution is 1 mL / min, wherein mobile phase A is water and mobile phase B is acetonitrile; the volume ratio of water to acetonitrile is 15:85. The detection wavelengths of the liquid chromatograph are 207 nm and 240 nm, and the flow rate of the mobile phase during the detection operation is 1 mL / min.

[0077] (3) Take 20 μL of the test solution and the reference solution respectively, inject them into the liquid chromatograph, record and observe the chromatogram, and calculate the related substances in dienolone acetate.

[0078] Example 2

[0079] The detection method of Example 1 was validated from the aspects of system applicability, destructive testing, peak purity, material balance investigation, destructive degradation impurities, quantitative limit, detection limit, linear relationship, injection precision, accuracy, intermediate precision, solution stability and durability.

[0080] 1. System suitability determination:

[0081] Take appropriate amounts of styrene acetate (SM), SM1-1 (styrene acetate starting material) and SM1-2 (styrene acetate intermediate), accurately weigh, dissolve in methanol and dilute to prepare stock solutions of each impurity containing about 0.5 mg of each impurity per 1 mL. Take appropriate amounts of each impurity stock solution and prepare each impurity location solution containing 10 μg of styrene acetate, SM1-1 and SM1-2 per 1 mL respectively. Add appropriate amounts of SM1-1 and SM1-2 stock solutions, and add appropriate amounts of styrene acetate to the same volumetric flask, dissolve in methanol and dilute to obtain a mixed solution of about 1 mg / mL of styrene acetate and 10 μg / mL of SM1-1 and SM1-2 as the system suitability solution.

[0082] 20 μL of each blank solution, dienol ketone acetate positioning solution, each impurity positioning solution and system suitability solution were injected into the high performance liquid chromatograph, and the detection wavelength was 207nm and 240nm. The chromatogram was recorded to examine the separation of each component. The results are shown in Tables 1 and 2 below. The system suitability spectrum is shown in Figure 5 and Figure 6 shown.

[0083] Table 1 Impurity location and separation - 207nm

[0084] Product Name Single Sample System Suitability Solutions Separation Blank solvent —— —— —— Dienolone acetate 10.337 10.270 9.6 SM1-1 23.817 23.553 1.5 SM1-2 33.017 32.783 11.0

[0085] Table 2 Impurity location and separation - 240nm

[0086] Product Name Single Sample System Suitability Solutions Separation Blank solvent —— —— —— Dienolone acetate 10.337 10.270 4.5

[0087] Table 1 shows the location and separation of impurities at 207nm, and Table 2 shows the location and separation of impurities at 240nm. It can be seen from the table that at a detection wavelength of 207nm, the peak order of the mixed solution is dienol ketone acetate, SM1-1, and SM1-2, and there is sufficient separation between dienol ketone acetate, SM1-1, and SM1-2, and the solvent peak and other impurities do not interfere with the determination. At a detection wavelength of 240nm, dienol ketone acetate has sufficient separation from other impurities, and the solvent peak does not interfere with the determination of dienol ketone acetate and other impurities.

[0088] Figure 5 This is the system suitability spectrum at 207nm. It can be seen from the figure that dienol ketone acetate, SM1-1 and SM1-2 can be effectively separated, and the blank solvent and other impurities do not interfere with the determination of dienol ketone acetate, SM1-1 and SM1-2.

[0089] Figure 6 This is the system suitability spectrum at 240nm. It can be seen from the figure that dienol ketone acetate and other impurities can be effectively separated, and the solvent peak does not interfere with the determination of dienol ketone acetate and other impurities.

[0090] 2. Destructive testing

[0091] Undestroyed: Take about 10 mg of dienol ketone acetate and place it in a 10 mL volumetric flask, add methanol to dissolve it to volume, mix well, and use it as the undestroyed solution.

[0092] Acid destruction: Take about 10 mg of dienol ketone acetate and place it in a 10 mL volumetric flask, add 1 mL of 0.1 mol / L hydrochloric acid solution, destroy it at 60°C for 24 hours, take it out, cool it at room temperature, add 1 mL of 0.1 mol / L sodium hydroxide solution to neutralize it, add methanol to dissolve it to the same volume, mix it well, and use it as the acid destruction solution.

[0093] Alkali destruction: Take about 10 mg of dienol ketone acetate and place it in a 10 mL volumetric flask, add 1 mL of 0.1 mol / L sodium hydroxide solution, destroy it at 60°C for 24 hours, take it out, cool it at room temperature, add 1 mL of 0.1 mol / L hydrochloric acid solution to neutralize it, add methanol to dissolve it to the same volume, mix it well, and use it as the alkaline destruction solution.

[0094] Oxidative destruction: Take about 10 mg of dienol ketone acetate and place it in a 10 mL volumetric flask, add 1 mL of 30% hydrogen peroxide solution, destroy it at 60°C for 2 hours, then take it out, cool it at room temperature, add methanol to dissolve it to the same volume, mix it well, and use it as the oxidative destruction solution.

[0095] Light destruction: Take about 10 mg of dienol ketone acetate and place it in a 10 mL volumetric flask, dissolve it in methanol to make up the volume, and mix it well. Light destruction is carried out at ultraviolet wavelengths of 254 nm and 365 nm, and samples are taken after 30 minutes as light destruction solutions.

[0096] High temperature destruction: Take about 10 mg of dienol ketone acetate and place it in a 10 mL volumetric flask. Destroy it at 100°C for 4 hours, then take it out and cool it at room temperature. Add methanol to dissolve it and mix it to make up the volume. This is the high temperature destruction solution.

[0097] Pyrolysis destruction: Take about 10 mg of dienol ketone acetate and place it in a 10 mL volumetric flask, add a small amount of methanol to dissolve it, destroy it at 100°C for 4 hours, then take it out, cool it at room temperature, add methanol to dissolve it to the same volume, mix it well, and use it as the pyrolysis destruction solution.

[0098] Take 20 μL of blank solution, system suitability solution and the above test solution and inject them into the chromatograph, record the chromatogram and analyze the changes of each impurity. It is concluded that the degradation products produced by the test sample under each destructive condition can be separated from the main peak and do not interfere with the determination of known impurities. The spectrum of each destructive test is as follows Figures 7 to 20 shown.

[0099] 3. Peak purity

[0100] The peak purity and spectral similarity of the test sample solutions under various destruction conditions were measured using a diode array detector. The results are shown in Table 3 below.

[0101] Table 3 Peak purity results

[0102] Destruction conditions Unbroken Acid damage Alkali damage Oxidative damage Light damage High temperature damage Pyrolysis Spectral purity 999 998 998 999 999 999 999

[0103] Table 3 shows the peak purity results. It can be seen from the table that SM1-1 and SM1-2 were not detected in dienol ketone acetate before and after destruction; the main peak spectral purity of the test sample under each destruction condition was not less than 990.

[0104] 4. Material balance investigation

[0105] The material balance was investigated based on the area normalization results. The results are shown in Tables 4 and 5 below.

[0106] Table 4 Material balance investigation results - 207nm

[0107]

[0108]

[0109] Table 5 Material balance investigation results - 240nm

[0110]

[0111] Table 4 shows the material balance investigation results -207nm, and Table 5 shows the material balance investigation results -240nm. It can be seen from the table that through the analysis of the sample material balance data, the degradation products produced by the test sample under various destruction conditions can be separated from the main peak, the purity of the main peak is not less than 990, and the degradation products do not interfere with the determination of known impurities; except for 60 minutes of illumination, the reduction in the destruction of the main peak of the test sample under other destruction conditions is basically consistent with the decrease in its content, and the total peak area detected after samples of the same concentration are destroyed under different conditions is basically similar, indicating that the chromatographic conditions can effectively detect degradation impurities.

[0112] 5. Destroy and degrade impurities

[0113] The failure conditions were the same as those for the destructive test, and the results are shown in Tables 6 and 7 below.

[0114] Table 6 Destructive test impurity spectrum (statistics by normalization method) -207nm

[0115]

[0116]

[0117]

[0118] Table 7 Destructive test impurity spectrum (statistics by normalization method) -240nm

[0119]

[0120]

[0121] Table 6 shows the impurity spectrum of the destructive test (statistics by normalization method) -207nm, Table 7 shows the impurity spectrum of the destructive test (statistics by normalization method) -240nm, Figure 7 It is the undestructed-destructive test spectrum at 207nm; Figure 8 It is the undestructed-destructive test spectrum at 240nm; Fig. 9It is the oxidation damage-destructive test spectrum at 207nm; Fig.10 It is the oxidation damage-destructive test spectrum at 240nm; Fig.11 It is the high temperature damage-destructive test spectrum at 207nm; Fig.12 It is the high temperature damage-destructive test spectrum at 240nm; Fig.13 It is the pyrolysis damage-destructive test spectrum at 207nm; Fig.14 It is the pyrolysis damage-destructive test spectrum at 240nm; Fig.15 It is the 207nm light damage-destructive test spectrum; Fig.16 It is the 240nm light damage-destructive test spectrum; Fig.17 It is the acid damage-destructive test spectrum at 207nm; Fig.18 It is the acid damage-destructive test spectrum at 240nm; Fig.19 It is the alkaline damage-destructive test spectrum at 207nm; Fig. 20 is a 240nm alkaline damage-destructive test spectrum; from Tables 6, 7 and Figures 7 to 20 It can be seen that the degradation products produced by the test sample under various destruction conditions can be separated from the main peak and do not interfere with the determination of known impurities. No SM1-1 and SM1-2 were detected for dienol ketone acetate before and after destruction; the spectral purity of the main peak of the test sample under various destruction conditions was not less than 990. The results of the material balance investigation showed that the reduction in the destruction of the main peak of the test sample under various destruction conditions was basically consistent with the decrease in its content. The total peak areas detected after the same concentration of samples destroyed under different conditions were basically similar, indicating that the chromatographic conditions can effectively detect degradation impurities.

[0122] 6. Limit of Quantitation and Limit of Detection

[0123] Take appropriate amount of dienol ketone acetate, SM1-1 and SM1-2, weigh accurately, dissolve in solvent and dilute to make each impurity stock solution containing about 0.5 mg of dienol ketone acetate and each impurity in 1 mL. Take appropriate amount of each impurity stock solution and make 1 mL of mixed solution containing 15 μg, 10 μg and 10 μg of dienol ketone acetate, SM1-1 and SM1-2 respectively. Use dilution method to measure its quantitative limit (S / N≥10) and detection limit (S / N≥3), and the results are shown in the table below. The results are shown in Tables 8 and 9 below.

[0124] Table 8 Quantitation limit results

[0125]

[0126] Table 9 Detection limit results

[0127]

[0128] Table 8 is the quantitative limit results, and Table 9 is the detection limit results. It can be seen from the table that under the chromatographic conditions of the present invention, the quantitative limits and detection limits of dienol ketone acetate, SM1-1 and SM1-2 meet the inspection requirements.

[0129] 7. Injection precision

[0130] A mixed solution containing 10 μg of each of dienol ketone acetate, SM1-1 and SM1-2 was prepared per 1 mL, and 20 μL was injected into the liquid chromatograph for 6 consecutive injections to record the peak area. The results are shown in Tables 10 and 11 below.

[0131] Table 10 Injection precision test results - 207nm

[0132]

[0133] Table 11 Injection precision test results - 240nm

[0134]

[0135]

[0136] Table 10 is the injection precision test result -207nm, and Table 11 is the injection precision test result -240nm. It can be seen from the table that after 6 consecutive injections, RSD is less than 2%, and the injection precision of enolone acetate, SM1-1 and SM1-2 is good.

[0137] 8. Solution stability test

[0138] 1) Stability test of reference solution

[0139] The injection precision samples were injected at 0h, 3h, 6h, 9h, 12h and 15h to investigate their intra-day stability. The results are shown in Table 12 below.

[0140] Table 12 Results of stability test of reference solution

[0141]

[0142] Table 12 shows the results of the stability test of the control solution. It can be seen from the table that dienol ketone acetate, SM1-1 and SM1-2 have good stability in the solvent within 15 hours.

[0143] 2) Test solution stability test

[0144] Take the test solution 1# under the repeatability item, inject it at 0h, 6h, 9h, 12h and 15h respectively, and calculate the impurity content by normalization method. The results are shown in Tables 13 and 14 below.

[0145] Table 13 Test solution stability test results (normalization method) -207nm

[0146]

[0147] Table 14 Test solution stability test results (normalization method) -240nm

[0148]

[0149] Table 13 shows the stability test results of the test solution (normalized method) -207nm, and Table 14 shows the stability test results of the test solution (normalized method) -240nm. It can be seen from the table that from the above measurement data, there was no obvious change in the test solution within 15 hours, and the test solution was relatively stable.

[0150] 9. Linearity and Range

[0151] Take appropriate amounts of dienol ketone acetate, SM1-1 and SM1-2, weigh accurately, dissolve in solvent and dilute to prepare each impurity stock solution containing about 0.5 mg of dienol ketone acetate and each impurity in 1 mL. Take appropriate amounts of each impurity stock solution and prepare 1 mL of mixed stock solutions containing 20 μg of dienol ketone acetate, SM1-1 and SM1-2 respectively as linear stock solutions. Take the above solutions and dilute them with solvents according to Table 15 below to prepare linear solutions of various concentrations.

[0152] Table 15 Preparation of linear solution

[0153]

[0154]

[0155] Accurately measure 20 μL of each of the above solutions and inject them into the high performance liquid chromatograph, record the chromatogram, determine the peak area, and perform linear regression with the peak area A as the ordinate and the concentration C as the abscissa. The results are shown in Tables 16 to 19 and Figures 1 to 4 shown.

[0156] Table 16 Dienolone acetate linear test results -207nm

[0157]

[0158] Table 17 Dienolone acetate linear test results - 240nm

[0159]

[0160]

[0161] Table 18 SM1-1 Linearity test results

[0162]

[0163] Table 19SM1-2 linear test results

[0164]

[0165] The calculation formula for the known impurity correction factor is as follows:

[0166] (f) Among them, k 米力农 : Slope of milrinone standard curve; k 杂质 : Impurity standard curve slope. The results are shown in Table 20 below.

[0167] Table 20 Correction factors for known impurities

[0168]

[0169]

[0170] The relative correction factors of all known impurities are in the range of 0.2 to 5, and the impurity content can be calculated by the self-control method with the correction factor added.

[0171] Table 20 shows the correction factors for known impurities. It can be seen from the table that the peak area shows a good linear relationship with the measured concentration for dienol ketone acetate in the range of 0.514 to 20.553 μg / mL, SM1-1 in the range of 0.484 to 19.366 μg / mL, and SM1-2 in the range of 0.480 to 19.185 μg / mL.

[0172] 10. Repeatability

[0173] Take the test sample of dienol ketone acetate and repeat the determination 6 times according to the relevant substance inspection method. The results are shown in Table 21 below.

[0174] Table 21 Repeatability test results

[0175]

[0176] Table 21 is the repeatability test results. It can be seen from the table that the sample was measured repeatedly 6 times, and SM1-1 and SM1-2 were not detected (n=6); the average value of the maximum unknown single impurity was 0.522%, and the RSD was 0.5% (n=6); the average value of total impurities was 1.280%, and the RSD was 0.9% (n=6). The repeatability of SM1-1 and SM1-2 was good.

[0177] 11. Intermediate precision

[0178] The same batch of samples were checked for related substances by the same operator at different times using the repeatability test method; the same batch of samples were checked for related substances by different operators at different times using the repeatability test method; the same batch of samples were checked for related substances by the same operator using different instruments at different times. The results are shown in Table 22 below.

[0179] Table 22 Intermediate precision test results

[0180]

[0181]

[0182] Table 22 is the intermediate precision test results. It can be seen from the table that under the chromatographic conditions, there is no obvious difference in the measurement results when the samples are tested by the same operator at different times; by different operators at different times; and by the same operator using different instruments at different times, and the intermediate precision is good.

[0183] 12. Accuracy (sample recovery rate)

[0184] Take appropriate amount of SM1-1 and SM1-2, weigh accurately, dissolve in methanol and dilute to make each impurity stock solution containing about 0.5 mg of each impurity per 1 mL. Take appropriate amount of each impurity stock solution and make 1 mL of mixed stock solution containing 20 μg of SM1-1 and SM1-2 respectively as recovery rate stock solution.

[0185] Accurately weigh an appropriate amount of enol ketone acetate, accurately pipette the above mixed stock solution into a solution containing about 1 mg / mL of enol ketone acetate and 50%, 100% and 200% of the limiting concentrations of SM1-1 and SM1-2 according to Table 23 below, and prepare 3 portions for each concentration.

[0186] Table 23 Preparation of recovery solution

[0187]

[0188] 20 μL of each of the above samples was injected, the peak area of ​​each known impurity was recorded, and the recovery rate and RSD of each known impurity were calculated. The results are shown in Tables 24 and 25 below.

[0189] Table 24SM1-1 sample recovery test results

[0190]

[0191]

[0192] Table 25SM1-2 sample recovery test results

[0193]

[0194] The calculation methods of each parameter are as follows:

[0195] Original amount = sample weight × impurity content;

[0196] Measured added amount = measured total amount - known amount;

[0197] Recovery rate = [measured added amount / added amount] × 100%.

[0198] Table 24 is the experimental results of the sample recovery rate of SM1-1, and Table 25 is the experimental results of the sample recovery rate of SM1-2. It can be seen from the table that the average sample recovery rate of SM1-1 is 100.7%, and the RSD value is 2.1%. The average sample recovery rate of SM1-2 is 99.7%, and the RSD value is 1.2%. The results are within an acceptable range.

[0199] 13. Durability

[0200] The durability of the method for related substances of dienol ketone acetate was verified mainly from four aspects: different proportions of mobile phase (±2%), different flow rates (±0.05mL / min), different column temperatures (±2℃) and different chromatographic columns. The results are shown in Tables 26 and 27 below. The durability spectrum is shown in Figures 21 to 34 shown.

[0201] Table 26 Durability test separation results

[0202]

[0203] Table 27 Durability test sampling results

[0204]

[0205]

[0206] Table 26 shows the separation results of the durability test, and Table 27 shows the sampling results of the durability test. Fig.21 It is 207nm-different flow rate 1-durability spectrum; Fig. 22 It is 240nm-different flow rate 1-durability spectrum; Fig.23 It is 207nm-different flow rate 2-durability spectrum; Fig.24 It is 240nm-different flow rate 2-durability spectrum; Fig.25 It is 207nm-different column temperature 1-durability spectrum; Fig.26 It is 240nm-different column temperature 1-durability spectrum; Fig. 27 It is 207nm-different column temperature 2-durability spectrum; Fig.28 It is 240nm-different column temperature 2-durability spectrum; Fig.29It is 207nm-different ratio 1-durability spectrum; Fig.30 It is 240nm-different ratio 1-durability spectrum; Fig.31 It is 207nm-different ratio 2-durability spectrum; Fig.32 It is 240nm-different ratio 2-durability map; Fig.33 It is 207nm-different chromatographic columns-durability spectrum; Fig.34 240nm-different chromatographic columns-durability spectrum; from Tables 26, 27 and Figures 21 to 34 It can be seen that the above durability test results show that the chromatographic conditions of this method can be fine-tuned by different proportions of mobile phase, different flow rates, different column temperatures and different chromatographic columns, which should have no obvious effect on the test results and has good durability.

[0207] The system applicability, destructive test, peak purity, material balance investigation, destructive degradation impurities, quantitative limit, detection limit, linear relationship, injection precision, accuracy, intermediate precision, solution stability and durability of the method for detecting related substances of dienol ketone acetate were verified, and the verification results were all within the acceptable range, indicating that the present invention can effectively detect related substances of dienol ketone acetate. According to the self-control method, the content of SM1-1 and SM1-2 does not exceed 1.0%, the maximum unknown single impurity does not exceed 1.5%, and the total impurity does not exceed 2.0%.

Claims

1. A method for determining related substances of dienolone acetate using HPLC, characterized in that: The specific steps are as follows: 1) Prepare reference solution and test solution; 2) Setting high performance liquid chromatography detection conditions; using a chromatographic column with octadecyl bonded silica as a filler; using a mobile phase as a mixed solution of mobile phase A and mobile phase B, and performing isocratic elution with the mobile phase, wherein mobile phase A is water and mobile phase B is acetonitrile; 3) Pipette the test solution and reference solution separately, inject them into the liquid chromatograph, record and observe the chromatogram, and calculate the related substances in dienol ketone acetate.

2. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 1), the preparation method of the test solution is: accurately weigh the dienol ketone acetate into a volumetric flask, and add methanol to dilute the volume to a solution containing 1 mg of dienol ketone acetate per 1 mL; the preparation method of the reference solution is: accurately weigh the test solution into a volumetric flask, and add methanol to dilute the volume to a solution containing 10 μg of dienol ketone acetate per 1 mL.

3. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 2), the chromatographic column is GL InertSustain C18, with a length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm.

4. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 2), the volume ratio of water to acetonitrile is 15:

85.

5. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 2), the column temperature of the chromatographic column is 35°C.

6. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 2), the flow rate of the mobile phase during the detection operation is 1 mL / min.

7. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 2), the flow rate of isocratic elution is 1 mL / min.

8. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 2), the isocratic elution time is 45 min.

9. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 3), the injection volume of the test solution and the reference solution was 20 μL.

10. The method for determining related substances of dienolone acetate by HPLC according to claim 1, characterized in that: In step 3), the detection wavelengths of the liquid chromatograph are 207 nm and 240 nm.

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