Method for separating and detecting related substances of milcidin hydrochloride tablets by high performance liquid chromatography

Abstract

The invention discloses a method for separating and detecting related substances of a milcidin hydrochloride tablet by high performance liquid chromatography, which comprises the following chromatographic conditions: a chromatographic column takes octadecylsilane chemically bonded silica as a filler, a phosphate buffer solution-acetonitrile solution as a mobile phase A and an acetonitrile-aqueous solution as a mobile phase B, the pH value of the phosphate buffer solution is 7.8-8.2, the column temperature is 20-30 DEG C, and the column temperature is 20-30 DEG C; the ratio of phosphate to acetonitrile in the mobile phase A is (90: 9)-(90: 11). According to the chromatographic column and the mobile phase system provided by the invention, the problems that an impurity G is overlapped with a main component and an impurity E is not separated from a main peak at a base line are solved, and other unknown impurities in a solvent, a blank auxiliary material and a test sample do not interfere with the detection of specific impurities; the separation degree between the impurity G and the impurity E and the separation degree between the main peak and the adjacent peak are both greater than 2.0, so that the impurities can be accurately and quantitatively analyzed, and the quality of the milcidin hydrochloride tablet can be effectively controlled.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and detecting related substances of mianserin hydrochloride tablets by high performance liquid chromatography. BACKGROUND

[0002] Mianserin hydrochloride tablets are a new generation of tetracyclic antidepressants developed and marketed by the Dutch Organon company. The pharmacological effect is to inhibit the presynaptic alpha adrenergic receptor, increase the concentration of norepinephrine in the synaptic cleft, and also has antagonistic effect on 5-HT2 receptors. It is mainly used for the treatment of depression with anxiety in clinical practice, and is widely used in the market due to its small cardiovascular toxicity, light anticholinergic adverse reactions, no interaction with antihypertensive drugs, etc. The product is a white film-coated tablet, which is white or white after removing the film coating.

[0003] The detection method is collected in the European Pharmacopoeia, the British Pharmacopoeia, and the Mianserin Hydrochloride Tablets Draft (2021). The European Pharmacopoeia uses thin layer chromatography to detect related substances, and the open system is easily affected by environmental factors such as humidity and temperature, and cannot accurately quantify known impurities and unknown impurities. The structures of impurities A, B, C, and E are disclosed in the above standards, and the detection method of related substances uses high-concentration ion pair reagent (sodium octane sulfonate). In the experiment, the instrument is difficult to balance and affects the column efficiency and service life of the chromatographic column.

[0004] According to the Technical Guidelines for Research on Impurities in Chemical Drugs, impurities can be divided into process impurities, degradation impurities, and impurities mixed from reactants and reagents, etc. according to their sources. Forced degradation test is very necessary for the separation degree investigation of unknown impurities. The purpose is mainly to provide important information about the separation of impurities (especially degradation products) and main components, sample stability and degradation pathways, etc. In order to investigate whether the method can effectively detect the degradation products in the raw material or preparation, suitable acid, base, light, heat, oxidation reaction, etc. Accelerated destructive test is used to verify the specificity of the analysis method according to the chemical structure characteristics of the drug, the prescription and process of the preparation, the storage condition, etc. SUMMARY

[0005] In the research process of the inventor, it is found that impurity E of mianserin hydrochloride raw material can be degraded to generate the following: ; In addition, the inventor also found that the degradation impurity G is generated by the following way: .

[0006] The impurity G is found to be the main degradation impurity in the forced degradation test, and it is necessary to study an analysis method capable of effectively detecting the degradation impurity in combination with the prescription process and possible degradation pathway analysis.

[0007] In view of the deficiencies of the prior art, the application provides a method for separating and detecting related substances of mianserin hydrochloride tablets by high performance liquid chromatography, which can effectively detect impurity E and impurity G and other impurities. The method for separating and detecting related substances of mianserin hydrochloride tablets by high performance liquid chromatography, the chromatographic conditions are that the chromatographic column is filled with octadecylsilane bonded silica gel, the mobile phase A is a phosphoric acid buffer-acetonitrile solution, the mobile phase B is an acetonitrile-water solution, the pH of the phosphoric acid buffer is 7.8-8.2, and the ratio of phosphoric acid to acetonitrile in the mobile phase A is 90:9-90:11.

[0008] Preferably, the related substances in the mianserin hydrochloride tablets include impurity G and impurity E, and the method can effectively detect impurity E and impurity G in the mianserin hydrochloride tablets.

[0009] Preferably, the chromatographic column is YMC Triart C18, 4.6mmx150mm, 3µm.

[0010] Preferably, the concentration of the phosphoric acid buffer is 0.018mol / L-0.022mol / L, preferably 0.02mol / L.

[0011] Preferably, the column temperature of the chromatographic column is 28-32℃, preferably 30℃.

[0012] Preferably, the flow rate of the mobile phase is 0.9-1.1ml / min.

[0013] Preferably, the gradient program of the mobile phase elution is as follows: 0-10 minutes, the volume percentage of the mobile phase A is kept unchanged at 80%, 10-25 minutes, the volume percentage of the mobile phase A is linearly changed from 80% to 40%, 25-40 minutes, the volume percentage of the mobile phase A is linearly changed from 40% to 25%, 40-45 minutes, the volume percentage of the mobile phase A is kept unchanged at 25%, 45-50 minutes, the volume percentage of the mobile phase A is linearly changed from 25% to 10%, 50-55 minutes, the volume percentage of the mobile phase A is kept unchanged at 10%, and then in the next 1 minute, the volume percentage of the mobile phase A is linearly changed to 80%, and then it is balanced.

[0014] Preferably, the detection wavelength is 250nm.

[0015] Preferably, the temperature of the sample injector is not higher than 10℃.

[0016] Further, a trapping column is installed between the mixer and the sample injector, and the disc is cph3040 4.0*30mm.

[0017] Preferably, the preparation of the sample is also included, comprising: Test solution: take the fine powder of the product, add solvent, and dissolve the methimazole hydrochloride by ultrasonic, dilute to the scale with solvent, prepare a solution with the concentration of 1mg / ml of methimazole hydrochloride, shake well, filter, and take the filtrate as the test solution; Control solution: accurately take the test solution, and dilute 200 times with solvent.

[0018] Preferably, the solvent is acetonitrile-water solution with a volume ratio of 50:50.

[0019] Further, the related substances in the methimazole hydrochloride tablets also include impurity A, impurity B and impurity C, the structure of impurity A is ; the structure of impurity B is ; and the structure of impurity C is .

[0020] Among them, in the bulk drug, impurity A, impurity B and impurity C are all collected in EP and BP, and impurity A (corresponding to impurity I) and impurity B (corresponding to impurity II) are collected in methimazole hydrochloride (draft). Combined with the synthesis process and the degradation pathway, whether impurities H, I, J and K have an influence on the detection of the concerned impurities is investigated.

[0021] Further, the related substances in the methimazole hydrochloride tablets also include impurity H, impurity I, impurity J and impurity K, the structure of impurity H is ; the structure of impurity I is ; the structure of impurity J is ; and the structure of impurity K is .

[0022] The method verification test proves that even if the above impurities in the raw material are transferred to the tablets in the production process, the above impurities will not affect the detection and quantification of impurities E and G; at the same time, the method provided by the present application can also realize the accurate detection of all the above impurities, and provides a quality control strategy guidance for the detection of related impurities of the bulk drug.

[0023] Compared with the prior art, the present application has the following beneficial effects: (1) The present application adopts a mobile phase system different from the prior art: solves the problem that the instrument is difficult to balance when using ion pair reagents under the conditions of the prior art, and prolongs the column efficiency and service life of the chromatographic column; effectively saves the cost.

[0024] (2) The chromatographic column and mobile phase system provided by the application solve the problems of overlapping of impurity G and main component and non-baseline separation of impurity E and main peak, and solvents, blank excipients, other unknown impurities in the test sample do not interfere with the detection of specific impurities; the separation degree between impurity G and impurity E and between the main peak and adjacent peaks is greater than 2.0, so that the impurities can be accurately quantitatively analyzed, and the quality of the misolimine hydrochloride tablets can be effectively controlled.

[0025] (3) The isocratic elution is replaced by gradient elution in the application, so that the two impurities (impurity G and impurity E) in the misolimine hydrochloride tablets and misolimine hydrochloride, blank excipients and impurities can be well separated and accurately quantitatively detected and analyzed, and the process impurities and degradation impurities (impurity A, impurity B, impurity C, impurity K, impurity H, impurity I and impurity J) in the misolimine hydrochloride raw material can be well separated from each other, impurity G, impurity E and misolimine hydrochloride, which indicates that the application has the detection ability for impurities in other raw materials. The quality of the misolimine hydrochloride tablets is more comprehensively controlled.

[0026] (4) The low-temperature injection method is adopted to effectively solve the problem of poor stability of impurity E solution at room temperature, which affects the accurate quantification of impurities. It is found that the solvent of impurity stock solution is more stable when methanol is used than when acetonitrile is used in the same mobile phase system, so it is determined that methanol is used to prepare the solvent of impurity stock solution, and then acetonitrile is used as the final solvent for dilution, so as to ensure the stability of the impurities.

[0027] (5) The problem of poor reproducibility of the peak shape of misolimine hydrochloride is solved by adding a guard column, and the service life of the chromatographic column is improved.

[0028] (6) The application compares the external standard method and the main component self-control method, and there is no difference between the two quantitative methods. The main component self-control method can be used for quantitative analysis of the two impurities, which saves the reference substance and saves the cost.

[0029] (7) The method of the application has strong specificity and applicability, high detection sensitivity, good linear relationship, high precision and accuracy, good durability of the detection method, high detection efficiency, and does not affect the accurate detection and quantification of impurities when the chromatographic conditions fluctuate to a certain extent, so that the quality of the product can be effectively controlled. BRIEF DESCRIPTION OF DRAWINGS

[0030] The drawings accompanying the specification of the application form a part of the application and serve to provide further understanding of the application, and the illustrative embodiments of the application and their descriptions serve to explain the application, and do not constitute improper limitations on the application.

[0031] Figure 1 The chromatogram of the system suitability test in Example 1.

[0032] Figure 2Chromatogram of the test solution of the batch of 2408921 used in Example 1.

[0033] Figure 3 Chromatogram of the test solution of the batch of 2408921 used in Example 2, which was exposed to light for 30 days.

[0034] Figure 4 Chromatogram of the specificity test in Example 2, in which the chromatogram from bottom to top is the positioning solution of impurity H (III-3), the positioning solution of impurity J (III-5), the positioning solution of impurity I (III-4), the positioning solution of impurity C, the positioning solution of impurity B, the positioning solution of impurity A, the positioning solution of impurity K (III-2), the positioning solution of impurity G, the positioning solution of impurity E, and the mixed solution.

[0035] Figure 5 Chromatogram under the method in Comparative Example 1, which is a reference to the method for related substances in BP2024 Mizoribine Hydrochloride Standard, in which the chromatogram from bottom to top is the solvent, the positioning solution of impurity G, and the spiked test solution.

[0036] Figure 6 Chromatogram under the method in Comparative Example 2, in which the chromatogram from bottom to top is the solvent, the blank excipient solution, the positioning solution of impurity B, the positioning solution of impurity A, the positioning solution of impurity G, the positioning solution of impurity E, the test solution, and the spiked test solution.

[0037] Figure 7 Comparison of the main peaks of the spiked test solution before and after the installation of the protective column in Comparative Example 4.

[0038] Figure 8 Comparison of the chromatographic peaks of Comparative Example 3 and Comparative Example 5 in Comparative Example 5.

[0039] Figure 9 Injection results under the ammonium formate mobile phase system in Comparative Example 6, in which the chromatogram from bottom to top is the blank solvent, the positioning solution of impurity G, the positioning solution of impurity E, and the spiked test solution.

[0040] Figure 10 Partial enlarged view of the spiked test solution in Comparative Example 6.

[0041] Figure 11 Injection results under the ammonium acetate mobile phase system in Comparative Example 7, in which the chromatogram from bottom to top is the blank solvent, the positioning solution of impurity G, the positioning solution of impurity E, and the spiked test solution.

[0042] Figure 12 Partial enlarged view of the spiked test solution in Comparative Example 7.

[0043] Figure 13The injection results of the ammonium formate mobile phase pH 8.0 system in Comparative Example 8, wherein the chromatogram from bottom to top is blank solvent, impurity G positioning solution, impurity E positioning solution, spiked sample solution.

[0044] Figure 14 The injection results of different buffer salt pH values in the mobile phase system of Example 1 in Comparative Example 9, wherein the chromatogram from bottom to top is spiked sample solution of buffer salt pH 6.0, buffer salt pH 6.5, buffer salt pH 7.0.

[0045] Figure 15 The PDA spectra of impurities G and E in the specificity test in Example 2. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0047] According to the synthesis route of the raw material of methimazole hydrochloride (as shown in the following formula), the generation path of each impurity is deduced as follows:

[0048] Impurity H: The intermediate III is reacted with 2-aminobenzyl alcohol of the previous step, and the product is continuously reacted to obtain impurity H, .

[0049] Impurity I: impurity B is hydrolyzed to generate: .

[0050] Impurity J: methimazole is reacted with dichloromethane to generate: .

[0051] Impurity K: the impurity generated by the intermediate chlorination is reacted with methimazole to generate: .

[0052] In the preparation of related substances, it is investigated whether all possible impurities will affect the detection of the impurities of interest.

[0053] Example 1, a method for separating and detecting related substances of methimazole hydrochloride tablets by high performance liquid chromatography Chromatographic conditions: The chromatographic column was filled with octadecylsilane bonded silica gel (YMC Triart C18, 4.6 mm × 150 mm, 3 μm or a column of equivalent performance is recommended); the mobile phase A was 20 mmol / L phosphate buffer (dissolve 2.64 g of diammonium hydrogen phosphate in 1000 ml of water and adjust the pH to 8.0 with phosphoric acid or ammonia)-acetonitrile (90:10), and the mobile phase B was acetonitrile-water (70:30). Linear gradient elution was performed according to the table below; the flow rate was 1.0 ml / min; the column temperature was 30°C; the detection wavelength was 250 nm; the injection volume was 10 μl; and the injector temperature was 10°C. A trapping column (Pahe cph3040 4.0×30 mm) was installed between the mixer and the injector, and a guard column (Yuexu Ultimate XB-C18 120A, 4.6 mm×10 mm, 5 μm) was installed in front of the chromatographic column.

[0054]

[0055] Solvent: water-acetonitrile (50:50).

[0056] Test solution: Take an appropriate amount of fine powder of this product (equivalent to 20 mg of miserine hydrochloride), place it in a 20 ml volumetric flask, add 15 ml of solvent, ultrasonicate for 10 minutes to dissolve the miserine hydrochloride, dilute to the scale with solvent, shake well, filter, and take the filtrate.

[0057] Control solution: Accurately measure 1 ml of the test solution, place it in a 200 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0058] Sensitivity solution: Take an appropriate amount of Miscellaneous Reference Standard, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing approximately 0.5 μg of Miscellaneous Hydrochloride per 1 ml.

[0059] System suitability solution: Take an appropriate amount of fine powder of this product (approximately equivalent to 20 mg of miserin hydrochloride), place it in a 20 ml volumetric flask, add 2 ml of 3% hydrogen peroxide solution, shake well to allow the fine powder to fully contact with 3% hydrogen peroxide, place in a 40°C water bath for 1 hour, take out, cool, dilute to the scale with solvent, shake well, filter, and take the filtrate.

[0060] System suitability requirements: In the system suitability solution chromatogram, impurity G and miserlin elute sequentially, and the theoretical plate number calculated based on the miserlin peak is not less than 5000; in the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be greater than 10.

[0061] Determination method: Accurately measure the test solution and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0062] Limit: If there are impurity peaks in the chromatogram of the test solution, deduct the peaks of excipients and solvent before the relative retention time of about 0.07, if there is a chromatographic peak consistent with the retention time of impurity G in the system suitability solution, the peak area after correction (multiplied by the correction factor 1.2) shall not be greater than the main peak area of the control solution (0.5%), the peak area of impurity E (relative retention time of about 0.67) shall not be greater than the main peak area of the control solution (0.5%), the peak area of other single impurity shall not be greater than the main peak area of the control solution (0.5%), the total amount of impurities shall not exceed 1.0%, and the chromatographic peak less than the main peak area of the sensitivity solution is ignored (0.05%).

[0063] Calculation formula:

[0064]

[0065] In the formula, A i is the peak area of impurities in the chromatogram of the test solution; A r is the peak area of misel in the chromatogram of the control solution; f is the correction factor of impurities, the correction factor of impurity G is 1.2, the correction factor of impurity E is 1.0, and other single impurities are calculated according to 1.0.

[0066] Test results: The chromatogram of the system suitability test is shown in Figure 1 , which meets the requirements of system suitability.

[0067] The test results of 2408921 batch of misel hydrochloride tablets at 0 days and naked tablet and blank tablet placed under light for 30 days are shown in Table 1, and the chromatogram is shown in Figures 2-3 .

[0068] Table 1 Test results of 2408921 batch of misel hydrochloride tablets at 0 days and naked tablet and blank tablet placed under light for 30 days

[0069] Naked tablet is unpackaged tablet, and blank tablet is uncoated tablet.

[0070] The light condition is: under the condition of light intensity 5000 lux, ultraviolet 85.0 μW / cm 2 , temperature 25℃±2℃, for about 30 days, so that the total illumination is not less than 3.6×10 6 lux·hr, and the near ultraviolet lamp energy is not less than 600W·hr / m 2 .

[0071] Example 2, method validation test Chromatographic conditions: the chromatographic conditions in Example 1 are used.

[0072] 2.1 System suitability test Solvent: water-acetonitrile (50:50).

[0073] Methimazole stock solution: about 4.4 mg of methimazole reference substance was taken in a 10 ml volumetric flask, dissolved and diluted to the mark with methanol, and shaken well.

[0074] The spiked sample solution prepared under the item of "2.3 Solution stability test" was taken as the system suitability solution.

[0075] Sensitivity solution: 1 ml of methimazole stock solution was taken in a 50 ml volumetric flask, diluted to the mark with solvent, shaken well, and 1 ml was taken in a 20 ml volumetric flask, diluted to the mark with solvent, and shaken well.

[0076] The solvent, sensitivity solution and system suitability solution (5 injections were repeated) were injected into the liquid chromatograph respectively, and the chromatogram was recorded. The RSD (%) of the retention time of each peak, the RSD (%) of the peak area, the theoretical plate number of the main peak in the first injection of the system suitability solution, the tailing factor, the signal-to-noise ratio and the separation degree were calculated.

[0077] Verification results: the RSD values of the retention time of impurity G, impurity E and the main peak were all less than 1% when the system suitability solution was injected 5 times repeatedly; the RSD values of the peak area were all less than 5%. In the chromatogram of the first injection of the system suitability solution, the theoretical plate number of the methimazole peak was 123789, which was greater than 5000; the separation degree between the peaks of impurity G and impurity E was greater than 2.0. In the chromatogram of the sensitivity solution, the signal-to-noise ratio of the main component peak height was 39, which was greater than 10. The results showed that the system suitability was good.

[0078] 2.2 Specificity test 2.2.1 Solvent and impurity interference test Impurity G stock solution: about 5 mg of impurity G reference substance was accurately weighed, placed in a 10 ml volumetric flask, dissolved and diluted to the mark with methanol, and shaken well.

[0079] Impurity E stock solution: about 5.7 mg of impurity E reference substance was accurately weighed, placed in a 10 ml volumetric flask, dissolved and diluted to the mark with methanol, and shaken well.

[0080] The sample solution prepared under the item of "2.3 Solution stability test" was taken.

[0081] The sample was prepared as follows, and the diluent or solvent was water-acetonitrile (50:50) unless otherwise specified.

[0082] 1) Impurity stock solution: 5 ml of impurity G stock solution and 5 ml of impurity E stock solution were taken into the same 50 ml; 2) Impurity A stock solution: Impurity A approximately 2 mg → 20 ml, diluent: methanol; 3) Impurity B reference substance stock solution: about 2 mg of Impurity B reference substance → 20 ml, dissolve in 5 ml of water, diluent: methanol; 4) Impurity C reference stock solution: Impurity C reference approximately 2 mg → 20 ml, diluent: methanol; 5) Impurity III-2 stock solution: Impurity III-2 approximately 2 mg → 10 ml, diluent: methanol; 6) Impurity III-3 reference substance stock solution: Impurity III-3 reference substance approximately 2 mg → 20 ml, diluent: methanol; 7) Impurity III-4 reference substance stock solution: Impurity III-4 reference substance approximately 2 mg → 10 ml, diluent: methanol; 8) Impurity III-5 reference substance stock solution: Impurity III-5 reference substance approximately 2 mg → 20 ml, diluent: methanol; 9) Impurity G localization solution: Impurity G reference stock solution 0.5ml → 50ml; 10) Impurity E localization solution: Impurity E reference stock solution 0.5ml → 50ml; 11) Impurity A location solution: Impurity A stock solution 1ml → 20ml; 12) Impurity B localization solution: Impurity B reference stock solution 1ml → 20ml; 13) Impurity C localization solution: Impurity C reference stock solution 1ml → 20ml; 14) Impurity III-2 (K) localization solution: Impurity III-2 stock solution 0.5ml → 20ml; 15) Impurity III-3 (H) localization solution: Impurity III-3 reference stock solution 1ml → 20ml; 16) Impurity III-4 (I) localization solution: Impurity III-4 reference stock solution 0.5 ml → 20 ml; 17) Impurity III-5 (J) localization solution: Impurity III-5 reference stock solution 1ml → 20ml; 18) Blank excipient solution: Add approximately 181 mg of blank excipient to 20 ml of solution, add 15 ml of solvent, sonicate for 10 minutes, dilute to volume with solvent, shake well, filter, and collect the filtrate; 19) Mixed solution: about 201 mg of fine powder of the product (about equivalent to 20 mg of mianserin hydrochloride) → 20 ml, add 8 ml of solvent, ultrasonic for 10 min, add impurity control stock solution 2 ml, impurity A stock solution 1 ml, impurity B control stock solution 1 ml, impurity C control stock solution 1 ml, impurity III-2 stock solution 0.5 ml, III-3 control stock solution 1 ml, III-4 control stock solution 0.5 ml and III-5 control stock solution 1 ml respectively in the same 20 ml volumetric flask, dilute to the mark with solvent, shake well, filter, take the filtrate.

[0083] Precisely measure the solvent, blank excipient solution, each impurity positioning solution, test sample solution and mixed solution, respectively inject into the liquid chromatograph, record the chromatogram.

[0084] Verification results: the results are shown in Figure 4 , the solvent and blank excipient solution do not interfere with the determination of impurity G, impurity E and each process impurity (impurity H, impurity K, impurity E, impurity I, impurity A, impurity J, impurity B, impurity C) and other impurities in the test sample solution which have been studied in mianserin hydrochloride raw materials. The separation degree between impurity G, impurity E and adjacent peaks in the mixed solution is greater than 1.5. The PDA spectrum of impurity G, E is shown in Figure 15 , the purity threshold angle of impurity G is 0.872, which is less than the purity threshold value 1.404, and the purity threshold angle of impurity E is 0.870, which is less than the purity threshold value 1.383, indicating that the spectral characteristics of impurity G and impurity E peaks are uniform, and there is no interference of impurities. It shows that the method is specific. The blank excipient and blank solvent are out of peak at relative retention time 0.07. When detecting impurities of related substances, it should be deducted.

[0085] 2.2.2 Forced degradation test Take mianserin hydrochloride tablets, self-made tablets and raw materials, and reference preparations for forced degradation test by the following method: Unbroken test sample solution: about 201 mg of fine powder of the product (about equivalent to 20 mg of mianserin hydrochloride) was accurately weighed and placed in a 20 ml volumetric flask, 15 ml of solvent was added, ultrasonic for 10 min, diluted to the mark with solvent, shake well, filter, discard the initial filtrate 5 ml, take the filtrate. The unbroken blank excipient solution and unbroken raw material test sample solution were prepared by the same method.

[0086] Acid-damaged sample solution: Take about 201 mg of fine powder of the product (about equivalent to 20 mg of mesnerline hydrochloride), accurately weigh and place in a top empty bottle, add 1 mol / L hydrochloric acid solution 2 ml, and place at room temperature for 24 h. Neutralize with 1 mol / L sodium hydroxide solution 2 ml, and transfer the total amount of 10 ml of solvent to a 20 ml volumetric flask. Ultrasonic for 10 min, dilute to the mark with solvent, shake well, filter, discard the first filtrate 5 ml, and take the subsequent filtrate. Prepare the acid-damaged blank excipient solution and the acid-damaged raw material sample solution by the same method.

[0087] Alkali-damaged sample solution: Take about 201 mg of fine powder of the product (about equivalent to 20 mg of mesnerline hydrochloride), accurately weigh and place in a top empty bottle, add 1 mol / L sodium hydroxide solution 2 ml, and place at room temperature for 24 h. Neutralize with 1 mol / L hydrochloric acid solution 2 ml, and transfer the total amount of 10 ml of solvent to a 20 ml volumetric flask. Process according to the method of "acid-damaged sample solution" to prepare the alkali-damaged blank excipient solution and the alkali-damaged raw material sample solution by the same method.

[0088] Oxidation-damaged sample solution: Take about 201 mg of fine powder of the product (about equivalent to 20 mg of mesnerline hydrochloride), accurately weigh and place in a top empty bottle, add 6% hydrogen peroxide solution 2 ml, and place in a 40°C water bath for 1 h. Take out, cool, transfer the total amount of 10 ml of solvent to a 20 ml volumetric flask, and process according to the method of "acid-damaged sample solution". Prepare the oxidation-damaged blank excipient solution and the oxidation-damaged raw material sample solution by the same method.

[0089] High-temperature-damaged sample solution: Take an appropriate amount of fine powder of the product, place it in an 80°C oven for 8 h, take it out, cool it, take about 201 mg (about equivalent to 20 mg of mesnerline hydrochloride), accurately weigh and place in a 20 ml volumetric flask, and process according to the method of "undamaged sample solution". Prepare the high-temperature-damaged blank excipient solution and the high-temperature-damaged raw material sample solution by the same method.

[0090] Light-damaged sample solution: Take an appropriate amount of fine powder of the product, place it in a weighing bottle, and place it in a light with an illuminance of 5000 lux and ultraviolet 85.0 μW / cm 2 for 10 days (total illuminance: 1.2 x 10 6 lux·hr, total ultraviolet energy: 204 W·hr / m 2 ), take about 201 mg (about equivalent to 20 mg of mesnerline hydrochloride), accurately weigh and place in a 20 ml volumetric flask, and process according to the method of "undamaged sample solution". Prepare the light-damaged blank excipient solution and the light-damaged raw material sample solution by the same method.

[0091] The precision of the solution was not destroyed and the destruction of the solution under various conditions, respectively, into the liquid chromatograph, record chromatogram. The test results of methsergide hydrochloride raw materials, methsergide hydrochloride tablets (self-made), methsergide hydrochloride tablets (reference preparation) are shown in Table 2.

[0092] The formula: degradation degree (%) = 100% - (A 破坏 / m 破坏 ) / (A 未破坏 / m 未破坏 ) x 100% Material conservation (%) = (A 破坏总 / m 破坏 ) / (A 未破坏总 / m 未破坏 ) x 100% A 破坏 : the main peak area in the destruction of the test solution chromatogram; m 破坏 : the sample weight of the product powder in the destruction of the test solution, mg; A 未破坏 : the main peak area in the non-destruction of the test solution chromatogram; m 未破坏 : the sample weight of the product powder in the non-destruction of the test solution, mg; A 破坏总 : the total peak area in the destruction of the test solution chromatogram; A 未破坏总 : the total peak area in the non-destruction of the test solution chromatogram.

[0093] Table 2 forced degradation test results

[0094] The verification results: in the above degradation test, the material conservation results of methsergide hydrochloride raw materials, methsergide hydrochloride tablets (self-made), methsergide hydrochloride tablets (reference preparation) under each destruction test condition are within 90%~110%, the degradation degree results are within 1.4%~7.1%, it can be seen that each destruction test condition is suitable; the purity angle of the main peak under each destruction test condition is less than the purity threshold, which shows that the peak purity of the main peak meets the requirements and does not contain other impurities which are not separated. Other peak purities also meet the requirements; according to the results, it can be seen that the raw material is relatively stable under acidic, alkaline, high temperature and light conditions; under the oxidation condition, the total impurities increase significantly from 0 to 2.26%, the main degradation impurities are unknown impurities with RRT 0.46 and impurity G, which are 0.19% and 2.07% respectively; under each degradation condition, the blank solvent peak after destruction and the degradation products do not interfere with the determination of impurity G, impurity E and other impurities in the test solution, and the separation degree between each specific impurity and the adjacent peak is greater than 1.5.

[0095] The analysis of the results of the destruction test showed that under each degradation condition, the destroyed blank excipient and the degradation products under each condition did not interfere with the determination of impurity G, impurity E and other impurities in the test solution. The degradation pathways of the self-made and reference preparations of mianserin hydrochloride tablets were basically the same. Both of them were stable under alkaline and high temperature conditions, and both of them were degraded under acidic, oxidative and light conditions, and the degradation impurities were basically the same under the same degradation conditions.

[0096] 2.3 Solution stability test Prepare the impurity reference stock solution according to the "2.2.1 Solvent and impurity interference test" item.

[0097] Control solution: accurately measure 1 ml of the test solution into a 200 ml volumetric flask, dilute to the mark with the solvent, shake well.

[0098] Test solution: take about 201 mg of fine powder of the product (about equivalent to 20 mg of mianserin hydrochloride), accurately weigh and place in a 20 ml volumetric flask, add 15 ml of solvent, ultrasonic for 10 min, dilute to the mark with the solvent, shake well, filter, discard the initial filtrate 5 ml, and take the subsequent filtrate.

[0099] Spiked test solution: take about 201 mg of fine powder of the product (about equivalent to 20 mg of mianserin hydrochloride), place in a 20 ml volumetric flask, add 15 ml of solvent, ultrasonic for 10 min, accurately measure 2 ml of the impurity reference stock solution into the same 20 ml volumetric flask, dilute to the mark with the solvent, shake well, filter, discard the initial filtrate 5 ml, and take the subsequent filtrate.

[0100] Control solution: accurately measure 1 ml of the test solution into a 200 ml volumetric flask, dilute to the mark with the solvent, shake well.

[0101] Place the impurity reference solution, control solution, test solution and spiked test solution at 10°C low temperature condition, respectively at 0h, 4h, 8h, 12h, 24h, 36h, 48h after preparation, sample analysis, record the chromatogram, and place the impurity reference stock solution at 2-8°C condition, re-dilute the control solution, investigate the stability of the impurity reference stock solution, calculate the percentage of the peak area of each component in the impurity reference solution chromatogram and the control solution chromatogram at each time point to the peak area at 0 hour, calculate the content of each impurity in the test solution and spiked test solution chromatogram at each time point by peak area normalization method.

[0102] The results of the verification: the control solution and the control solution were placed at 10°C for 48 hours, and the peak area ratio of each component peak at each observation time point to the peak area at 0h was within the range of 92%-105%, indicating that the control solution and the control solution were stable at 10°C for at least 48 hours; the control solution was prepared after the control solution was placed at 2-8°C for 68 hours, and the peak area percentage of impurities G and impurities E to the peak area at 0h was within the range of 92%-105%, indicating that the control solution was stable at 2-8°C for at least 68 hours.

[0103] The absolute value of the difference between the content of each known impurity and other single impurity at each time point in the chromatogram of the test solution and the spiked test solution and the content at 0h was less than 0.05%, the absolute value of the difference between the total impurity content and the content at 0h was less than 0.05%, and no new other degradation impurities greater than the reporting limit were produced, indicating that the spiked test solution was stable at 10°C for at least 48 hours.

[0104] 2.4 Quantitative limit and detection limit test Take the impurity E positioning solution and the impurity G positioning solution prepared under the "2.2.1 Solvent and impurity interference test" item.

[0105] Take the Millipore stock solution prepared under the "2.1 System suitability test" item.

[0106] Misce Lin stock solution: accurately measure 1 ml of misce Lin stock solution, place it in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0107] Quantitative limit solution: accurately measure 3 ml of misce Lin quantitative limit stock solution, impurity G positioning solution and impurity E positioning solution, respectively, in the same 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0108] Detection limit solution: accurately measure 3 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0109] Prepare 6 portions of quantitative limit concentration solution in parallel, inject 1 time for detection limit, and record the chromatogram. Report the minimum quantitative concentration and detection concentration (μg / ml), quantitative limit and detection limit (ng), percentage equivalent to limit concentration, percentage equivalent to test sample concentration and RSD of peak area of each component peak in 6 quantitative limit tests, respectively.

[0110] The results of the verification are as follows: the minimum quantitative concentrations of impurity G, impurity E and mescaline hydrochloride are 0.15 μg / ml, 0.15 μg / ml and 0.30 μg / ml, respectively, which are equivalent to 0.02%, 0.02% and 0.03% of the concentration of the test sample, respectively, all less than 0.05% of the concentration of the test sample, equivalent to 3.0%, 3.0% and 6.0% of the limit concentration, respectively, all less than 10% of the limit concentration, and the signal-to-noise ratios of the peak heights of impurity G, impurity E and mescaline are 14, 20 and 20, respectively, in the quantitative limit test; the minimum detection concentrations of impurity G, impurity E and mescaline hydrochloride are 0.045 μg / ml, 0.045 μg / ml and 0.089 μg / ml, respectively, which are equivalent to 0.005%, 0.005% and 0.009% of the concentration of the test sample, respectively, all less than 0.025% of the concentration of the test sample, equivalent to 0.90%, 0.90% and 1.8% of the limit concentration, respectively, all less than 5% of the limit concentration, and the signal-to-noise ratios of the peak heights of impurity G, impurity E and mescaline are 4, 5 and 6, respectively, in the detection limit test; the RSD values of the peak areas of impurity G, impurity E and mescaline in the quantitative limit reproducibility test are 1.4%, 4.2% and 1.5%, respectively, all less than 10%, indicating good reproducibility of the quantitative limit.

[0111] 2.5 Linearity and range test The mescaline reference substance stock solution prepared under "2.1 System suitability test" was taken.

[0112] The impurity reference substance stock solution prepared under "2.2.1 Solvent and impurity interference test" was taken.

[0113] Linear stock solution: 2 ml of the impurity G reference substance stock solution, 2 ml of the impurity E reference substance stock solution and 2 ml of the mescaline reference substance stock solution were accurately measured into the same 20 ml volumetric flask, diluted to the mark with the solvent and shaken well.

[0114] Linear solution 1 (quantitative limit solution): the first solution in the quantitative limit reproducibility test was used as the first point of linearity.

[0115] Linear solutions 2-7: 1 ml, 1 ml, 2 ml, 2 ml, 3 ml and 2 ml of the linear stock solution were accurately measured into 50 ml, 20 ml, 25 ml, 20 ml, 20 ml and 10 ml volumetric flasks, respectively, diluted to the mark with the solvent and shaken well. They were used as the linear solutions of 20%, 50%, 80%, 100%, 150% and 200% limit concentrations.

[0116] The above linear solutions were accurately measured and injected into the liquid chromatograph, respectively, and the chromatograms were recorded. The concentration C (μg / ml) of impurity G, impurity E and mescaline hydrochloride was taken as the abscissa, and the peak area A of each component was taken as the ordinate, and linear regression was performed.

[0117] The verification results are as follows: the impurity G in the range of 0.1486 μg / ml to 9.9039 μg / ml, the concentration C and the peak area A show a good linear relationship, the linear regression equation is A=8193.0643C-283.7653, the correlation coefficient r is 0.9999, greater than 0.99, the absolute value of the Y-axis intercept and the percentage of the 100% concentration response value is 0.70%, less than 25%. The impurity E in the range of 0.1485 μg / ml to 9.8991 μg / ml, the concentration C and the peak area A show a good linear relationship, the linear regression equation is A=9677.0957C-148.2420, the correlation coefficient r is 0.9999, greater than 0.99, the absolute value of the Y-axis intercept and the percentage of the 100% concentration response value is 0.31%, less than 25%. The concentration C of the mescaline hydrochloride in the range of 0.2979 μg / ml to 9.9291 μg / ml, the peak area A of the mescaline and the peak area A of the mescaline hydrochloride show a good linear relationship, the linear regression equation is A=9697.8028C-140.9008, the correlation coefficient r is 0.9999, greater than 0.99, the absolute value of the Y-axis intercept and the percentage of the 100% concentration response value is 0.29%, less than 25%.

[0118] 2.6 Correction factor The impurity control stock solution prepared under "2.2.1 Solvent and impurity interference test" was taken.

[0119] The mescaline hydrochloride control stock solution: about 4.4 mg of mescaline control (about equivalent to 5 mg of mescaline hydrochloride) was accurately weighed into a 10 ml volumetric flask, dissolved and diluted to the mark with the solvent, and shaken well.

[0120] The correction factor stock solution: 2 ml of the impurity G control stock solution, 2 ml of the impurity E control stock solution, and 2 ml of the mescaline hydrochloride control stock solution were accurately measured into the same 20 ml volumetric flask, diluted to the mark with the solvent, and shaken well.

[0121] The correction factor solution 1: 1 ml, 2 ml, and 3 ml of the correction factor stock solution were accurately measured into three 20 ml volumetric flasks, diluted to the mark with the solvent, and shaken well as the correction factor solution.

[0122] The above correction factor solution was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The relative correction factor was calculated by the ratio of "main component peak area / concentration" to "impurity peak area / concentration" in the three concentration mixed solutions. Personnel 1 calculated the linear solution 3, the linear solution 4, and the linear solution 5 under the "linear and range test" test, and the average of the three relative correction factors was taken as the correction factor. The average of the correction factors of the two persons was taken as the final correction factor.

[0123] Verification result: The correction factor of impurity G was 1.2 and the standard deviation was 0.5%, within 10%; the correction factor of impurity E was 1.0 and the standard deviation was 1.0%, within 10%, meeting the requirements. The correction factor of impurity G was determined as 1.2 and the correction factor of impurity E was determined as 1.0.

[0124] 2.7 Precision test 2.7.1 Reproducibility test Take the impurity reference substance stock solution prepared under "2.2.1 Solvent and impurity interference test".

[0125] Take the sensitivity solution prepared under "2.1 System suitability test".

[0126] Impurity reference solution: accurately measure 2 ml of the reference substance stock solution into a 20 ml volumetric flask, dilute to the mark with the solvent, and shake well.

[0127] System suitability solution: take about 201 mg of fine powder of the product (about equivalent to 20 mg of mesnerine hydrochloride), add 2 ml of 3% hydrogen peroxide solution into a 20 ml volumetric flask, and place in a 40°C water bath for 1 hour, take out, cool, dilute to the mark with the solvent, shake well, filter, discard the first 5 ml of the filtrate, and take the subsequent filtrate.

[0128] Spiked sample solution: prepare the spiked sample solution according to "2.3 Solution stability test". Prepare 6 samples in parallel.

[0129] Control solution: accurately measure 1 ml of each spiked sample solution into 6 200 ml volumetric flasks, respectively, dilute to the mark with the solvent, and shake well.

[0130] Accurately measure the solvent, system suitability solution, impurity reference solution, control solution, and spiked sample solution, respectively, inject into the liquid chromatograph, and record the chromatogram. Calculate the content of known impurities according to the impurity reference external standard method and the principal component self-control method with correction factor, calculate the content of other single impurities according to the principal component self-control method, and calculate the total amount of impurities.

[0131] Verification result: prepare 6 spiked sample solutions in parallel, calculate the content of impurity G and impurity E in the spiked sample solution according to the external standard method, the RSD values are 0.4% and 0.3% respectively, both less than 3%; calculate the content of impurity G and impurity E in the spiked sample solution according to the principal component self-control method with correction factor, the RSD values are 1.1% and 1.1% respectively, and the RSD value of the total amount of impurities is 1.1%, all less than 3%. The above results show that the method has good reproducibility.

[0132] The difference between the content of each specific impurity calculated by the external standard method and the content of the specific impurity calculated by the principal component self-control method with correction factor, and the ratio of the impurity content calculated by the external standard method, are all within ±10% of the external standard method determination results, and there is no significant difference. The results show that the principal component self-control method with correction factor can accurately quantify the content of each known impurity.

[0133] 2.7.2 Intermediate precision test Under the reference of repetitive test method, the sensitivity solution, system suitability solution, 6 spiked sample solutions and control solution were prepared by the same method at different times by different analysts on different liquid chromatographs, and the content of each specific impurity was calculated by the principal component self-control method with correction factor. The content of other single impurity was calculated by the principal component self-control method, and the total impurity was calculated. The intermediate precision of the method was investigated by combining the results of 12 repetitive tests.

[0134] Verification results: On different dates, different personnel prepared sensitivity solution, system suitability solution, 6 spiked sample solutions and control solution according to the operation steps of repetitive test, and determined the content of impurities G and E by the principal component self-control method with correction factor according to the repetitive test method on different liquid chromatographs. The RSDs were 0.7% and 0.6% respectively, both less than 3%. Other single impurities were not detected, and the RSD of the total impurity was 0.7%, less than 3%. Combined with the results of 6 repetitive tests, a total of 12 results were combined to calculate the content of impurities G and E, and the RSDs were 3.3% and 2.8% respectively, both less than 6%. The RSD of the total impurity was 3.1%, less than 6%, indicating that the precision of the method was good.

[0135] 2.8 Accuracy test Take the control solution prepared under "2.2.1 Solvent and impurity interference test".

[0136] Take the impurity control solution prepared under "2.7.1 Reproducibility test".

[0137] Sample solution: take about 201 mg of fine powder of the product (about equivalent to 20 mg of mesnerine hydrochloride), add 15 ml of solvent to a 20 ml volumetric flask, ultrasonic for 10 min, dilute to the mark with solvent, shake well, filter, discard the first 5 ml of filtrate, and take the subsequent filtrate.

[0138] Each concentration recovery solution: about 201 mg of fine powder of the product (about equivalent to 20 mg of methimazole hydrochloride) was taken into a 20 ml volumetric flask, 15 ml of solvent was added, and ultrasonic treatment was performed for 10 min. 1, 2, and 3 ml of the mixed reference substance stock solution was precisely measured and added into three 20 ml volumetric flasks containing the fine powder, respectively, and diluted to the mark with the solvent, and shaken well. The initial filtrate was discarded, and the subsequent filtrate was collected. Each concentration recovery solution was prepared in triplicate.

[0139] The solvent, impurity reference substance solution, test sample solution, and each recovery solution were precisely measured and injected into the liquid chromatograph, respectively. The chromatogram was recorded, and the measured amount of each specific impurity was calculated by the peak area according to the impurity reference substance external standard method, and the recovery rate was calculated. Verification results: within the concentration range of 50% to 150%, the average recoveries of impurities G and E were 100.4% and 102.7%, respectively, both within the range of 90% to 108%, indicating that the method has high accuracy.

[0140] 2.9 Durability test The impurity reference substance stock solution was prepared according to the "2.2.1 Solvent and impurity interference test" item.

[0141] The sensitivity solution was prepared according to the "2.1 System suitability test" item.

[0142] System suitability solution: prepared according to the system suitability solution preparation method in Example 1. Blank excipient solution: prepared according to the "2.2.1 Solvent and impurity interference test" item in Example 2. Spiked test sample solution: prepared according to the "2.3 Solution stability test" item in Example 2. Control solution: 1 ml of the spiked test sample solution was precisely measured and placed in a 200 ml volumetric flask, and diluted to the mark with the solvent, and shaken well.

[0143] Under each condition, the blank excipient solution, sensitivity solution, system suitability solution, spiked test sample solution, and control solution were precisely measured and injected into the liquid chromatograph, respectively, and the chromatogram was recorded. The content of each known impurity and other single impurity in the spiked test sample solution was calculated, and the total amount of impurities was calculated. The chromatographic column used was YMC Triart C18, different batches 20426 and 21235. The results are shown in Table 3.

[0144] Table 3 Durability test results of different condition changes on the determination results of related substances of the sample

[0145] Verification results: under each condition, the theoretical plate number calculated according to the Mescalin peak in the system suitability solution chromatogram was greater than 5000; in the system repeatability test, the RSD value of the main peak retention time was less than 1%, and the RSD value of the peak area was less than 2%; the signal-to-noise ratio of the main component peak in the sensitivity solution chromatogram was greater than 10. The absolute value of the difference between the content of each impurity in the spiked test sample solution and the standard condition was less than 20% of the detection result of the standard condition. It is shown that the method is better in durability within the range of 28-32℃ of column temperature, 0.9-1.1ml / min of flow rate, 7.8-8.2 of pH value of mobile phase, 90:9-90:11 of mobile phase ratio, 0.018-0.022mol / L of buffer salt concentration of mobile phase, and replacement of chromatographic column of the same manufacturer, same specification and same type but different batches.

[0146] Reference Example 1, reference to BP2024 Mescalin Hydrochloride Standard Impurity Method.

[0147] Solvent: mobile phase.

[0148] Each impurity reference solution: an appropriate amount of each reference substance was weighed, dissolved and diluted with solvent to prepare impurity B 0.1mg / ml, impurity A 0.1mg / ml, impurity G 0.05mg / ml, impurity E 0.1mg / ml solution.

[0149] Spiked test sample solution: about 200mg of fine powder of the product (about equivalent to 20mg of Mescalin Hydrochloride) was taken and placed in a 20ml volumetric flask, an appropriate amount of solvent was added, and ultrasonic was performed for 10min. Each impurity reference solution was 1ml, which was placed in the same 20ml volumetric flask, diluted to the mark with solvent, shaken well, and filtered. The filtrate was taken.

[0150] Chromatographic conditions: octane-bonded silica gel was used as the filler (Waters Symmetry C8, 3.9mm x 150mm, 5µm, code: LCC231111); pH 3.0 sodium octane sulfonate buffer (sodium octane sulfonate 5.0g, water 350ml, dissolved, pH adjusted to 3.0 with phosphoric acid, water added to 400ml, shaken well)-methanol (37:63) was used as the mobile phase; the flow rate was 0.5ml / min; the column temperature was 30℃; the detection wavelength was 250nm; the injection volume was 10µl.

[0151] The solvent, spiked test sample solution and impurity G positioning solution were precisely measured and injected into the liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 1. Figure 5 According to the impurity G positioning solution, it was judged that impurity G in the spiked test sample solution was coincided with the main peak, and this method was not applicable.

[0152] Reference Example 2, on the basis of Reference Example 1, gradient was added and the chromatographic column was replaced Take the stock solutions of each impurity reference substance under "Comparative Example 1".

[0153] Impurity location solution: Measure 1 ml of each impurity reference stock solution, place it in four 20 ml volumetric flasks, dilute to the scale with solvent, and shake well.

[0154] Blank excipient solution: Take about 180 mg of blank excipient, place it in a 20 ml volumetric flask, add appropriate amount of solvent, sonicate for 10 minutes, dilute to the scale with solvent, shake well, filter, and take the filtrate.

[0155] Test solution: Take about 200 mg of the fine powder of this product (equivalent to 20 mg of nicarbazine hydrochloride), place it in a 20 ml volumetric flask, add appropriate amount of solvent, sonicate for 10 minutes, dilute to the scale with solvent, shake well, filter, and take the filtrate.

[0156] Impurity G positioning solution and spiked test solution Take the impurity G positioning solution and spiked test solution under Comparative Example 1.

[0157] Chromatographic conditions: Octadecylsilane bonded silica gel was used as the filler (YMC Triart C18, 4.6 mm × 150 mm, 3 µm, code: LCC221203); pH 3.0 sodium octane sulfonate buffer (5.0 g of sodium octane sulfonate was dissolved in 350 ml of water, the pH was adjusted to 3.0 with phosphoric acid, and water was added to 400 ml, and shaken)-methanol (90:10) was used as the mobile phase A; methanol-water (90:10) was used as the mobile phase B, and gradient elution was performed according to the table below; the flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 250 nm; and the injection volume was 10 µl.

[0158]

[0159] Accurately measure the solvent, blank excipient solution, test solution, each impurity location solution and spiked test solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0160] The results are as follows Figure 6 As shown in the figure, impurity G and impurity E did not achieve baseline separation, and the solvent peak interfered with the detection of impurity E. The reason for this was that the high concentration of the ion pair reagent and the gradient elution procedure in the chromatographic conditions caused large baseline fluctuations and were difficult to balance.

[0161] Comparative Example 3: Comparative Test of the Effect of Solvents on Stability Impurity B reference substance stock solution <1>: about 2 mg of Impurity B reference substance was taken in a 20-ml volumetric flask, about 2 ml of water was added, dissolved and diluted to the mark with acetonitrile, and shaken well. Impurity B reference substance stock solution <2>: about 2 mg of Impurity B reference substance was taken in a 20-ml volumetric flask, about 2 ml of water was added, dissolved and diluted to the mark with methanol, and shaken well. Impurity A reference substance stock solution: a solution with a concentration of about 0.1 mg / ml was prepared by dissolving and diluting with acetonitrile. Impurity G reference substance stock solution: a solution with a concentration of about 0.1 mg / ml was prepared by dissolving and diluting with acetonitrile. Impurity E reference substance stock solution <1>: a solution with a concentration of about 0.1 mg / ml was prepared by dissolving and diluting with acetonitrile. Impurity E reference substance stock solution <2>: a solution with a concentration of about 0.1 mg / ml was prepared by dissolving and diluting with methanol.

[0162] Reference solution: 1 ml of Impurity G reference substance stock solution and 1 ml of Impurity E reference substance stock solution <1> were taken in the same 20-ml volumetric flask, diluted to the mark with the solvent, and shaken well.

[0163] Impurity B and Impurity E mixed solution: 1 ml of Impurity B reference substance stock solution <2> and 1 ml of Impurity E reference substance stock solution <2> were taken in the same 20-ml volumetric flask, diluted to the mark with the solvent, and shaken well. (The solution was divided into two parts, one was placed at room temperature and the other was placed in the refrigerator for cold storage, for investigating the low-temperature stability of the solution) Spiked sample solution: about 200 mg of fine powder of the product (about equivalent to 20 mg of mesnerine hydrochloride) was taken in a 20-ml volumetric flask, an appropriate amount of solvent was added, and ultrasonic treatment was performed for 10 min. 1 ml of Impurity B reference substance stock solution <1>, 1 ml of Impurity A reference substance stock solution, 1 ml of Impurity G reference substance stock solution, and 1 ml of Impurity E reference substance stock solution <1> were taken in the same 20-ml volumetric flask, diluted to the mark with the solvent, shaken well, filtered, and the filtrate was taken.

[0164] Reference solution: 1 ml of Impurity G reference substance stock solution and 1 ml of Impurity E reference substance stock solution <1> were taken in the same 20-ml volumetric flask, diluted to the mark with the solvent, and shaken well.

[0165] Chromatographic conditions: Except that the injection port was 5°C and no guard column was installed before the chromatographic column, and the gradient elution program had the following parameters, the other chromatographic conditions were the same as in Example 1.

[0166]

[0167] The reference solution, the Impurity B and Impurity E mixed solution, and the Impurity B and Impurity E mixed solution placed in the refrigerator for cold storage were precisely taken and injected into the liquid chromatograph at different time points, and the chromatogram was recorded. The results are shown in Table 4.

[0168] Table 4 Stability of reference solution (stock solution solvent: acetonitrile), Impurity B and Impurity E mixed solution (stock solution solvent: methanol) room temperature, low temperature stability comparison results

[0169] The results show that: under room temperature conditions, when the stock solution solvent of the reference standard is acetonitrile, impurity G is stable for at least 12 hours, and impurity E is stable within 2 hours; when the stock solution solvent of the reference standard is methanol, impurity E is stable within 4 hours, so when the stock solution solvent is methanol, impurity E has better stability. Under low temperature conditions, impurities B and E are stable for at least 18 hours. In summary, when the stock solution solvent is methanol and under low temperature conditions, it is beneficial to the storage of impurity E.

[0170] Comparative Example 4, comparison of results of low temperature injection and comparison of effects of guard column A guard column (Ultimate XB-C18 120A, 4.6mm x 10mm, 5μm) was installed before the chromatographic column, the sample injector temperature was 5℃, and the rest of the chromatographic conditions were the same as those of Comparative Example 3.

[0171] The spiked test sample solution and the control solution were placed at a low temperature of 5℃, and were injected and analyzed at 0h, 2h, 4h, 8h, 12h after preparation, respectively. The chromatograms were recorded, and the results are shown in Table 5. The comparison chart and results of the spiked test sample solution before and after the installation of the guard column are shown in Table 6 and Figure 7 .

[0172] Table 5 Comparison of chromatographic peak areas under low temperature injection

[0173] Table 6 Comparison of chromatographic peak areas before and after installation of the guard column

[0174] The results show that: under low temperature conditions of 5℃, the spiked test sample solution and the reference standard solution are stable for at least 12 hours. After installing the guard column, the peak height of each impurity and the main peak of the same spiked test sample solution is higher when injected on the same chromatographic column multiple times, and the peak shape and response are significantly improved and the chromatographic peak shape has good repeatability.

[0175] The reason for analyzing the effect of installing the guard column on the chromatographic peak is that the excipients commonly used in the preparation may accumulate at the end of the column, causing the peak shape to gradually deteriorate. Therefore, a guard column (Ultimate XB-C18 120A, 4.6mm x 10mm, 5μm) was installed before the column, which greatly improved the deterioration of the peak shape.

[0176] Comparative Example 5, comparison of effects of gradient elution program Prepare the reference standard solution with the same concentration according to the "Solution stability" item.

[0177] The rising rate of the mobile phase B in the gradient program was found to be too fast (1 min) when the percentage was increased from 75% to 90% in the later sample detection, resulting in a steep gradient peak and affecting the detection of impurities. After comprehensive consideration, the rising program was slowed down under the original conditions, and the temperature of the sample injector was investigated at 10°C, with the rest of the conditions remaining unchanged.

[0178] The chromatographic conditions were the same as those in Example 1.

[0179] The elution gradient of the sample in Comparative Example 3 and Comparative Example 5 was compared as shown in Figure 8 The gradient peak in Comparative Example 5 was slow, which did not interfere with the detection of each impurity; the temperature of the sample injector was 10°C, and the reference solution was stable for at least 17 hours. Therefore, the temperature of the sample injector was set to 10°C, and this method was used for the determination of the related substances of the methsuximil hydrochloride tablets.

[0180] In Comparative Example 6, the mobile phase system of the ammonium formate buffer Reference to the impurity reference solution and the positioning solution prepared under the item of “2.2.1 Solvent and Impurity Interference Test” Reference solution: accurately measure 1 ml of impurity E reference solution and 1 ml of impurity G reference solution in the same 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0181] Spiked sample solution: take about 200 mg of fine powder of the product (about equivalent to 20 mg of methsuximil hydrochloride), add an appropriate amount of solvent to a 20 ml volumetric flask, sonicate for 10 min, accurately measure 2 ml of the reference solution into the same 20 ml volumetric flask, dilute to the mark with solvent, shake well, filter, and take the filtrate.

[0182] Chromatographic conditions: octadecylsilane-bonded silica gel (YMC Triart C18, 4.6 mm x 150 mm, 3 µm) as the filler; 20 mmol / L ammonium formate buffer salt solution-acetonitrile (90:10) as the mobile phase A; acetonitrile-water (70:30) as the mobile phase B, and the other chromatographic conditions were the same as those in Example 1. Accurately measure the blank solvent, impurity G positioning solution, impurity E positioning solution, and spiked sample solution, and inject them into the liquid chromatograph, respectively, to record the chromatogram. The test results are shown in Figure 9 and Figure 10 .

[0183] The results show that under this mobile phase system, the separation degree of impurity G and impurity E is low, and there is an unknown impurity peak between impurity G and impurity E, which does not achieve baseline separation, affecting the detection of impurity G and impurity E; the baseline separation between the main component peak and the previous adjacent impurity peak is not achieved, affecting the detection of impurities; and the baseline fluctuation under this system causes the solvent peak to be generated, affecting the detection of unknown impurities. Therefore, the ammonium formate system is not suitable for the determination of the related substances of methsuximil hydrochloride tablets.

[0184] Comparative Example 7, ammonium acetate buffer mobile phase system Each solution was taken from under the item of "Comparative Example 6".

[0185] The chromatographic conditions were as follows: 20 mmol / L ammonium acetate buffer salt solution-acetonitrile (90:10) as mobile phase A; acetonitrile-water (70:30) as mobile phase B, and other chromatographic conditions were the same as in Example 1. The blank solvent, impurity G positioning solution, impurity E positioning solution, and spiked test sample solution were precisely measured, respectively injected into the liquid chromatograph, and the chromatogram was recorded. The test results are shown in Table 1. Figure 11 and Figure 12 .

[0186] The results showed that: under this mobile phase system, there were solvent peaks brought in by the system before and after impurity E, which were similar to impurity E, affecting the determination of impurity E; the main component peak and the adjacent impurity peak after the main component peak did not reach baseline separation, affecting the detection of unknown impurities. Therefore, the ammonium acetate system is not suitable for the determination of related substances of misolimbine hydrochloride tablets.

[0187] Comparative Example 8, under the chromatographic conditions of Comparative Example 6, the pH value of the mobile phase was adjusted to 8.0, and other chromatographic conditions were unchanged.

[0188] The blank solvent, impurity G positioning solution, impurity E positioning solution, and spiked test sample solution were precisely measured, respectively injected into the liquid chromatograph, and the chromatogram was recorded. The test results are shown in Table 1. Figure 13 .

[0189] The results showed that: on the basis of Comparative Example 6, the pH value of the buffer salt in mobile phase A was adjusted to 8.0 in the present technology, under this mobile phase system, the separation degree of impurity G and impurity E increased, indicating that the pH value of 8.0 in the present technology has the advantage of increasing the separation degree of known impurities, but the ammonium formate system introduces more solvent peaks, which interfere with the detection of unknown impurities, and the unknown impurities adjacent to the main peak before and after the main peak are not detected, which should be coincided with the main peak, still not suitable for the determination of related substances of misolimbine hydrochloride tablets.

[0190] Comparative Example 9, pH value of buffer salt endurance limit Impurity E reference substance stock solution: about 1.1 mg of impurity E reference substance was weighed into a 10 ml volumetric flask, dissolved and diluted to the mark with methanol, and shaken well. Impurity G reference substance stock solution: about 1 mg of impurity G reference substance was weighed into a 10 ml volumetric flask, dissolved and diluted to the mark with methanol, and shaken well.

[0191] Spiked test sample solution: prepare the spiked test sample solution according to the item of "2.3 Solution stability test".

[0192] The chromatographic conditions were the same as in Example 1. The standard sample solution was precisely measured, and the mobile phase of different pH buffer salt (pH 7.0, 6.5, 6.0 respectively adjusted by phosphoric acid) was injected into the liquid chromatograph, and the chromatogram was recorded. The test results are shown in Table 1. Figure 14 .

[0193] The results show that when the pH value of the buffer salt is 6.0, impurity G and impurity E are completely coincident, when the pH value of the buffer salt is 6.5, the separation degree of impurity G and impurity E is 4.7, and when the pH value of the buffer salt is 7.0, the separation degree of impurity G and impurity E is 15.9, which indicates that with the increase of the pH value of the buffer salt, the separation degree of impurity G and impurity E increases. Combined with the pH value tolerance of the chromatographic column and the pH value of 0.02M diammonium hydrogen phosphate itself is about 8.0, which is convenient for operation, and the separation degree of the process impurities of the raw material drug, therefore, 20mmol / L diammonium hydrogen phosphate buffer salt solution (pH 8.0) is the optimal choice.

Claims

1. A method for separating and detecting related substances of Miserin Hydrochloride Tablets by high performance liquid chromatography, characterized in that, The chromatographic conditions were as follows: the column was filled with octadecylsilane bonded silica gel, mobile phase A was phosphate buffer-acetonitrile solution, mobile phase B was acetonitrile-water solution, the pH of the phosphate buffer was 7.8-8.2, and the ratio of phosphate buffer to acetonitrile in mobile phase A was 90:9-90:11; Related substances in Miserin Hydrochloride Tablets include impurities G and E. The structural formula of impurity E is ; The structural formula of impurity G is .

2. the method for separating and detecting Miserin Hydrochloride Tablets related substances by high performance liquid chromatography according to claim 1, is characterized in that, The chromatographic column is YMC Triart C18, 4.6 mm × 150 mm, 3 μm.

3. the method for separating and detecting Miserin Hydrochloride Tablets related substances by high performance liquid chromatography according to claim 1, is characterized in that, The concentration of phosphate buffer is 0.018 mol / L to 0.022 mol / L, preferably 0.02 mol / L.

4. the method for separating and detecting Miserin Hydrochloride Tablets related substances by high performance liquid chromatography according to claim 1, is characterized in that, The column temperature is 28~32℃, preferably 30℃.

5. the method for separating and detecting Miserin Hydrochloride Tablets related substances by high performance liquid chromatography according to claim 1, is characterized in that, The gradient program of mobile phase elution was: From 0 to 10 minutes, the volume percentage of mobile phase A remained constant at 80%. From 10 to 25 minutes, the volume percentage of mobile phase A linearly increased from 80% to 40%. From 25 to 40 minutes, the volume percentage of mobile phase A linearly increased from 40% to 25%. From 40 to 45 minutes, the volume percentage of mobile phase A remained constant at 25%. From 45 to 50 minutes, the volume percentage of mobile phase A linearly increased from 25% to 10%. From 50 to 55 minutes, the volume percentage of mobile phase A remained constant at 10%. Then, within 1 minute, the volume percentage of mobile phase A linearly increased to 80%, and then reached equilibrium.

6. the method for separating and detecting Miserin Hydrochloride Tablets related substances by high performance liquid chromatography according to claim 1, is characterized in that, The detection wavelength is 250 nm.

7. the method for separating and detecting the related substance of Miserin Hydrochloride Tablets by high performance liquid chromatography according to claim 1 is characterized in that, The injector temperature was no higher than 10°C.

8. the method for separating and detecting the related substance of Miserin Hydrochloride Tablets by high performance liquid chromatography according to claim 1 is characterized in that, A trapping column was installed between the mixer and the injector, and the disc was cph3040 4.0×30mm.

9. the method for separating and detecting the related substance of Miserin Hydrochloride Tablet by high performance liquid chromatography according to claim 1 is characterized in that, It also covers sample preparation, including: Test solution: Take an appropriate amount of fine powder of this product, place it in a container, add solvent, sonicate to dissolve the hydrochloric acid methylcellulose, dilute to the scale with solvent to prepare a solution with a concentration of 1 mg / ml of hydrochloric acid methylcellulose, shake well, filter, and take the filtrate as the test solution; Control solution: Accurately measure the test solution and dilute it 200 times with solvent.

10. The method for separating and detecting the related substances of Miserin Hydrochloride Tablets by high performance liquid chromatography according to claim 9, wherein The solvent was an acetonitrile-water solution with a volume ratio of 50:50.

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