Method for analyzing impurity spectrum of melogabalin besylate bulk drug

Abstract

The invention relates to the technical field of medicine detection, in particular to a melogabalin besylate bulk drug impurity spectrum analysis method, which adopts a high performance liquid chromatography method of adding a chiral inclusion agent into a mobile phase for determination, and liquid chromatography conditions are as follows: the mobile phase A comprises a phosphate-chiral inclusion agent buffer solution; the mobile phase B comprises acetonitrile and water, and a gradient elution mode is adopted; the flow velocity is 0.5 to 2.0 mL / min; a chromatographic column stationary phase filling agent is achiral silane bonded silica gel; the column temperature is 15-55 DEG C; and the detection wavelength is 190 nm to 230 nm. The method has the advantages of simplicity in operation, low cost, good selectivity, high accuracy and strong universality, is suitable for research on the impurity spectrum of melogabalin besylate bulk drugs so as to effectively control the product quality, and can be expanded for research on impurity spectrums of other amino acids and similar drugs thereof.

Description

Technical Field

[0001] The invention relates to an analysis method for the impurity spectrum of melogabalin besylate raw material medicine, belonging to the technical field of medical detection. Background Art

[0002] Mirogabalin Besilate is a new drug developed by Daiichi Sankyo of Japan for the treatment of peripheral neuropathic pain (PNP). Peripheral neuropathic pain is caused by peripheral nerve damage or dysfunction caused by various reasons. Typical PNP includes diabetic and postherpetic neuralgia. Mirogabalin Besilate is mainly used for the treatment of diabetic peripheral neuropathy, postherpetic neuralgia and fibromyalgia. Compared with gabapentin and pregabalin, which are currently used to treat neuralgia on the market, mirogabalin besilate has unique binding properties and long-lasting effects with voltage-dependent calcium channels. Clinical studies have shown that the efficacy of merogabalin besilate is significantly higher than that of gabapentin and pregabalin. At the same time, merogabalin besilate has the advantages of high safety and good tolerability, and is developing into a world-class drug for the treatment of neuralgia.

[0003] Impurity spectrum research is a crucial link in the quality research process of raw materials, and it is also the focus of current drug regulatory authorities in various countries. How to effectively and reasonably study and control the sources and destinations of raw materials, reaction by-products, impurities introduced by equipment, and degradation-induced impurities involved in the production process of raw materials is a top priority for pharmaceutical companies and research institutions in drug research and development.

[0004] Genotoxic impurities can cause irreversible damage to human DNA, so drug regulatory authorities in various countries have a strict management attitude towards the residual amount of such impurities in drugs. At present, the pharmaceutical industry is also very cautious in the research of such impurities. Genotoxic impurities can cause serious side effects even in trace amounts, so the residual limit in drugs is usually controlled at a low level (ppm level or lower) to ensure the safety of patients' medication.

[0005] The structural formula of melogabalin besylate (CAS No.: 1138245-21-2) is: The patent application with publication number CN118420477A discloses the synthesis route of melogabalin besylate. The impurity spectrum information involved is as follows: .

[0006] From the data in the above table, we can see that: (1) Melogabalin besylate has multiple chiral centers and unsaturated bonds at the same time. During the synthesis and storage process, various types of isomers (enantiomers, diastereomers, double bond position isomers) impurities will be generated. Among them, different diastereomeric impurities are enantiomeric, and different double bond position isomers are cis-trans isomers. (2) Melogabalin besylate is a benzenesulfonate salt. The solvent ethanol is used in its synthesis process, which may introduce genotoxic impurities of benzenesulfonate esters.

[0007] Because of the high similarity between the structures and similar physical and chemical properties, isomeric impurities are usually difficult to separate from the main components or specific impurities, especially enantiomeric impurities, which cannot be separated at all using conventional non-chiral filler chromatographic columns. At present, the separation of isomeric impurities of melogabalin besylate in the industry mostly uses expensive chiral stationary phase chromatographic columns for separation and analysis. For example, the technologies disclosed in the patent applications with publication numbers CN104755456A (original research), CN104245951A (original research), CN113740471A, CN117630229A, and CN117388402A are all separations performed on chiral stationary phase chromatographic columns, which further increases the testing cost. In addition, due to the gap between the performance of chiral stationary phase fillers and the performance of conventional reversed-phase chromatographic column fillers with a higher degree of maturity, the peak shapes of each isomer are not ideal under most conditions, and there is currently no reported analytical method that can effectively separate the above-mentioned isomeric impurities and process impurities at the same time.

[0008] When the chromatographic peak shape cannot achieve the ideal Gaussian distribution shape, the separation between trace residual impurities and the target component peak cannot be displayed, resulting in failure to meet the requirements of pharmaceutical quality control regulations, which has become a major problem that plagues analysts in the industry.

[0009] For genotoxic impurities with lower control limits, conventional UV detectors usually cannot meet the sensitivity requirements, and expensive mass spectrometers are often used for control studies. Currently, there are no reports on the detection methods of genotoxic impurities in melogabalin besylate raw materials.

[0010] How to study the impurity spectrum of melogabalin besylate raw material simply, accurately and at low cost has become an urgent problem to be solved. Summary of the invention

[0011] The present invention aims at the deficiencies in the prior art and provides an analytical method for the impurity spectrum of melogabalin besylate raw material drug. The method has the advantages of simple operation, low cost, good selectivity, high accuracy and strong universality, is suitable for the study of the impurity spectrum of melogabalin besylate raw material drug, and thus effectively controls the product quality, and can be expanded to be used for the study of the impurity spectrum of other amino acids and similar drugs thereof.

[0012] The technical solution of the present invention to solve the above technical problems is as follows: a method for analyzing the impurity spectrum of melogabalin besylate raw material drug, the analysis method is: S1. Preparation of solution: Preparation of blank solvent: Mix acetonitrile and water evenly to obtain blank solvent; Preparation of test solution: Accurately weigh the melogabalin besylate bulk drug substance to be tested, dissolve it in blank solvent and dilute it to make a test solution containing 4.0 mg per 1 ml; Preparation of control solution: accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add blank solvent to dilute to the scale, and shake well; accurately measure 1 mL of the solution in the 100 mL volumetric flask, place it in a 10 mL volumetric flask, add blank solvent to dilute to the scale, and shake well to obtain the control solution; S2. Use liquid chromatography to detect the test solution and the control solution: The detector of the liquid chromatography is an ultraviolet detector, and the chromatographic conditions are: mobile phase A includes phosphate-chiral inclusion agent buffer, mobile phase B includes acetonitrile and water, and gradient elution is adopted; the flow rate is 0.5-2.0 mL / min; the chromatographic column stationary phase filler is non-chiral silane bonded silica gel; the column temperature is 15°C-55°C; and the detection wavelength is 190nm-230nm.

[0013] Furthermore, the chiral inclusion agent is at least one of cyclodextrin, crown ether, vancomycin, and norvancomycin.

[0014] Further, the pH value of the mobile phase A is 2.0-8.8; the phosphate concentration in the phosphate-chiral inclusion agent buffer of the mobile phase A is 1-200 mmol / L; the chiral inclusion agent concentration in the phosphate-chiral inclusion agent buffer is 1-200 mmol / L; The mobile phase A contains acetonitrile or does not contain acetonitrile, and the volume ratio of the phosphate-chiral inclusion agent buffer to acetonitrile in the mobile phase A is (6:4) to (10:0).

[0015] Preferably, the pH value of the mobile phase A is 3.0-4.0; the phosphate concentration in the phosphate-chiral inclusion agent buffer of the mobile phase A is 70-90 mmol / L; the chiral inclusion agent concentration in the phosphate-chiral inclusion agent buffer is 80-120 mmol / L; The volume ratio of the phosphate-chiral inclusion agent buffer to acetonitrile in the mobile phase A is (8:2) to (9.5:0.5).

[0016] Furthermore, the mobile phase B contains methanol or does not contain methanol, and the volume ratio of acetonitrile, water and methanol in the mobile phase B is (4:6:0) to (8:1:1).

[0017] Preferably, the volume ratio of acetonitrile, water and methanol in the mobile phase B is 6:4:0.5.

[0018] Furthermore, the achiral alkyl group bonded to the silica gel in the chromatographic column stationary phase filler is selected from any one of octadecyl, octyl, phenyl, phenylhexyl, pentafluorophenyl, and octadecyl-pentafluorophenyl.

[0019] Preferably, the flow rate is 0.8-1.5 mL / min; the column temperature is 20° C.-30° C.; and the detection wavelength is 205 nm-215 nm.

[0020] Further, the gradient elution is: 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%; 2) When the elution time is 10 min, the volume fraction of mobile phase A is 100%; 3) When the elution time is 35 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; 4) When the elution time is 55 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%.

[0021] Furthermore, in the blank solvent, the volume ratio of acetonitrile to water is 2:8.

[0022] The beneficial effects of the present invention are: 1. The present invention uses the chiral inclusion composite method for a comprehensive study of the impurity spectrum of melogabalin besylate, which involves not only chiral isomer impurities, but also residual raw materials, achiral isomer impurities, genotoxic impurities, degradation impurities and other process by-products.

[0023] 2. By adding inexpensive chiral inclusion agents, such as cyclodextrin, to the mobile phase, the inclusion effect of the internal cavity structure of cyclodextrin on isomers is utilized to form chiral inclusions with different physical and chemical properties, which produce different distribution coefficients in the mobile phase and the stationary phase. Various types of isomeric impurities (including enantiomeric impurities) can be effectively completely baseline separated, and even trace amounts of impurities can meet the separation requirements. At the same time, the use of expensive chiral chromatographic columns is avoided, solving the problems of isomeric impurities such as "difficult separation, difficult quantification of trace components, and poor regulatory compliance" while significantly reducing the testing cost. It can also provide a technical basis for the separation of isomeric impurities in other compounds.

[0024] 3. The ultraviolet absorption of melogabalin besylate is weak and terminal absorption, and the selection of additives is very limited. Cyclodextrin has a lower ultraviolet absorption cutoff wavelength and basically no ultraviolet absorption, and has the advantages of rapid and stable inclusion complexation. It does not interfere with the determination of target impurities and the results have good repeatability and accuracy.

[0025] 4. The present invention uses conventional detectors to effectively monitor the genotoxic impurity ethyl benzenesulfonate in the melogabalin benzenesulfonate raw material (the detection limit can be as low as 10 ppm), avoiding the use of expensive mass spectrometry detectors, reducing detection costs, and expanding the universality of the method.

[0026] 5. The present invention adopts high performance liquid chromatography, which does not require complicated pretreatment, has low cost, strong universality, high precision, good reproducibility, has a strong stability indication effect, and can be smoothly transferred to different laboratories for quality control of melogabalin besylate raw materials, thereby ensuring its safety, effectiveness and quality controllability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the blank solution chromatogram in Example 1; Figure 2 It is the chromatogram of the mixed solution in Example 1; Figure 3 The chromatogram of the test solution in Example 1; Figure 4 is the chromatogram of the control solution in Example 1; Figure 5 It is the chromatogram of the mixed solution in Example 2; Figure 6 The chromatogram of the spiked test solution in Example 3; Figure 7 The chromatogram of the spiked test solution in Example 4; Figure 8 This is the chromatogram of the spiked test solution in Example 5; Fig. 9 This is the chromatogram of the test solution (acid degradation) in Example 7; Fig.10 This is the chromatogram of the test solution (alkaline degradation) in Example 8; Fig.11 This is the chromatogram of the test solution (oxidative degradation) in Example 9; Fig.12 This is the chromatogram of the test solution in Comparative Example 1; Fig.13 This is the solution chromatogram of the system suitability in Comparative Example 2; Fig.14 This is the chromatogram of the test solution in Comparative Example 2. DETAILED DESCRIPTION

[0028] The specific implementation of the present invention is described in detail below. The present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used are only for describing specific embodiments and are not intended to limit the present invention.

[0030] An analytical method for the impurity spectrum of melogabalin besylate raw material drug, the analytical method comprising: S1. Preparation of solution: Preparation of blank solvent: Mix acetonitrile and water evenly to obtain blank solvent; Preparation of test solution: Accurately weigh the melogabalin besylate bulk drug substance to be tested, dissolve it in blank solvent and dilute it to make a test solution containing 4.0 mg per 1 mL; Preparation of control solution: accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add blank solvent to dilute to the scale, and shake well; accurately measure 1 mL of the solution in the 100 mL volumetric flask, place it in a 10 mL volumetric flask, add blank solvent to dilute to the scale, and shake well to obtain the control solution; S2. Use liquid chromatography to detect the test solution and the control solution: The detector of the liquid chromatography is an ultraviolet detector, and the chromatographic conditions are: mobile phase A includes phosphate-chiral inclusion agent buffer, mobile phase B includes acetonitrile and water, and gradient elution is adopted; the flow rate is 0.5-2.0 mL / min; the chromatographic column stationary phase filler is non-chiral silane bonded silica gel; the column temperature is 15°C-55°C; and the detection wavelength is 190nm-230nm.

[0031] Specifically, the chiral inclusion agent is at least one of cyclodextrin, crown ether, vancomycin, and norvancomycin.

[0032] Specifically, the pH value of the mobile phase A is 2.0-8.8; the phosphate concentration in the phosphate-chiral inclusion agent buffer of the mobile phase A is 1-200 mmol / L; the chiral inclusion agent concentration in the phosphate-chiral inclusion agent buffer is 1-200 mmol / L; The mobile phase A contains acetonitrile or does not contain acetonitrile, and the volume ratio of the phosphate-chiral inclusion agent buffer to acetonitrile in the mobile phase A is (6:4) to (10:0).

[0033] Preferably, the pH value of the mobile phase A is 3.0-4.0; the phosphate concentration in the phosphate-chiral inclusion agent buffer of the mobile phase A is 70-90 mmol / L; the chiral inclusion agent concentration in the phosphate-chiral inclusion agent buffer is 80-120 mmol / L; The volume ratio of the phosphate-chiral inclusion agent buffer to acetonitrile in the mobile phase A is (8:2) to (9.5:0.5).

[0034] The phosphate-chiral inclusion agent buffer is an aqueous solution prepared with phosphate and a chiral inclusion agent, and is adjusted to a suitable pH with phosphoric acid.

[0035] Specifically, the mobile phase B contains methanol or does not contain methanol, and the volume ratio of acetonitrile, water and methanol in the mobile phase B is (4:6:0) to (8:1:1).

[0036] Preferably, the volume ratio of acetonitrile, water and methanol in the mobile phase B is 6:4:0.5.

[0037] Specifically, the achiral alkyl group bonded to the silica gel in the chromatographic column stationary phase filler is selected from any one of octadecyl, octyl, phenyl, phenylhexyl, pentafluorophenyl, and octadecyl-pentafluorophenyl.

[0038] Preferably, the non-chiral alkyl group bonded to the silica gel in the chromatographic column stationary phase filler is a phenyl group.

[0039] Preferably, the flow rate is 0.8-1.5 mL / min; the column temperature is 20° C.-30° C.; and the detection wavelength is 205 nm-215 nm.

[0040] Specifically, the gradient elution is: 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%; 2) When the elution time is 10 min, the volume fraction of mobile phase A is 100%; 3) When the elution time is 35 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; 4) When the elution time is 55 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%.

[0041] Specifically, in the blank solvent, the volume ratio of acetonitrile to water is 2:8.

[0042] More specifically, the chromatographic column is selected from any one of ZORBAX Phenyl, Eclipse Plus Phenyl-Hexyl, and ACE Excel3 C18-PFP.

[0043] During the test, the control solution and the test solution in step S1 were accurately measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded. The impurity content was calculated according to the melogabalin self-control method, and the calculation formula was: Impurity content (%) = As / Ar × 0.1% Where: As—the peak area of ​​each impurity in the test solution; Ar—main peak area of ​​the reference solution.

[0044] 1. Detection Example Example 1 (1) Solution preparation: Blank solvent: a solution prepared from acetonitrile and water (the volume ratio of acetonitrile to water is 2:8).

[0045] Preparation of test solution: Take an appropriate amount of the product (melogabalin besylate raw material to be tested), accurately weigh it, add blank solvent to dissolve it and dilute it to make a solution containing 4.0 mg of the product per 1 mL.

[0046] Preparation of control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add blank solvent to dilute to the scale, and shake well; Accurately measure 1 mL of the above solution, place it in a 10 mL volumetric flask, add blank solvent to dilute to the scale, and shake well to obtain the control solution.

[0047] Mixed solution: Take appropriate amount of melogabalin besylate reference substance, impurity 1 reference substance, impurity 2 reference substance, impurity 3 reference substance, impurity 4 reference substance, impurity 5 reference substance, impurity 6 reference substance and impurity 7 reference substance, add appropriate amount of solvent to dissolve, and take 1 mL into a 100 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0048] (2) Put the prepared solution into liquid chromatography for testing: The liquid chromatography conditions were: The chromatographic column was ZORBAX Phenyl (4.6×150mm, 5μm); the mobile phase A was prepared by mixing phosphate-chiral inclusion agent buffer and acetonitrile in a volume ratio of 9:1, wherein the concentration of potassium hydrogen phosphate in the phosphate-chiral inclusion agent buffer was 80 mmol / L, the concentration of cyclodextrin was 100 mmol / L, and the pH of the phosphate-chiral inclusion agent buffer was adjusted to 3.5 with phosphoric acid; the mobile phase B was prepared by mixing acetonitrile, water, and methanol in a volume ratio of 6:4:0.5; the detection wavelength was 210nm; the flow rate was 1.2mL / min; the column temperature was 25℃; the injection volume was 5μL; and the detector was an ultraviolet detector.

[0049] The elution gradient is: 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%; 2) When the elution time is 10 min, the volume fraction of mobile phase A is 100%; 3) When the elution time is 35 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; 4) When the elution time is 55 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; Test results such as Figure 1-Figure 4 ,from Figure 1-Figure 4 It can be seen that the separation between melogabalin (peak time is about 13 minutes, melogabalin besylate will have two peaks in the liquid chromatography system, namely benzenesulfonic acid peak and melogabalin peak, of which the melogabalin peak is the active ingredient peak of the drug, and the area of ​​the peak is usually used as the quantitative parameter of the drug) and each impurity meets the requirements. Impurity 1 and impurity 2 can meet the separation requirements, and the separation between other known impurities can meet the separation requirements.

[0050] Example 2 (1) Solution preparation: Blank solvent: a solution prepared from acetonitrile and water (the volume ratio of acetonitrile to water is 2:8).

[0051] Mixed solution: Take appropriate amount of melogabalin besylate reference substance, impurity 1 reference substance, impurity 2 reference substance, impurity 3 reference substance, impurity 4 reference substance, impurity 5 reference substance, impurity 6 reference substance and impurity 7 reference substance, add appropriate amount of solvent to dissolve, and take 1 mL into a 100 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0052] (2) Put the prepared solution into liquid chromatography for testing: The liquid chromatography conditions were: The chromatographic column was Eclipse Plus Phenyl-Hexyl (4.6×150mm, 4μm); the mobile phase A was prepared by using phosphate-chiral inclusion agent buffer and acetonitrile in a volume ratio of 9.5:0.5, wherein the concentration of potassium dihydrogen phosphate in the phosphate-chiral inclusion agent buffer was 70 mmol / L, the concentration of crown ether was 120 mmol / L, and the pH of the phosphate-chiral inclusion agent buffer was adjusted to 4 with phosphoric acid; the mobile phase B was prepared by using acetonitrile, water, and methanol in a volume ratio of 8:1:1; the detection wavelength was 205nm; the flow rate was 0.8mL / min; the column temperature was 20℃; the injection volume was 5μL; and the detector was an ultraviolet detector.

[0053] The elution gradient is: 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%; 2) When the elution time is 10 min, the volume fraction of mobile phase A is 100%; 3) When the elution time is 35 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; 4) When the elution time is 55 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; Test results such as Figure 5 ,from Figure 5 It can be seen that the separation between melogabalin (peak time is about 12 minutes) and various impurities meets the requirements.

[0054] Example 3 (1) Solution preparation: Blank solvent: a solution prepared from acetonitrile and water (the volume ratio of acetonitrile to water is 2:8).

[0055] Preparation of test solution: Take an appropriate amount of the product (melogabalin besylate raw material to be tested), accurately weigh it, add blank solvent to dissolve it and dilute it to make a solution containing 4.0 mg of the product per 1 mL.

[0056] Preparation of control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add blank solvent to dilute to the scale, and shake well; Accurately measure 1 mL of the above solution, place it in a 10 mL volumetric flask, add blank solvent to dilute to the scale, and shake well to obtain the control solution.

[0057] Spiked test solution: Take appropriate amount of this product (melogabalin besylate API to be tested), impurity 1 reference substance, impurity 2 reference substance, impurity 3 reference substance, impurity 4 reference substance, impurity 5 reference substance, impurity 6 reference substance and impurity 7 reference substance, add appropriate amount of solvent to dissolve, and take 1mL into a 100mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0058] (2) Put the prepared solution into liquid chromatography for testing: The liquid chromatography conditions were: The chromatographic column was ACE Excel 3 C18-PFP (4.6×150mm, 3μm); the mobile phase A was prepared by mixing phosphate-chiral inclusion agent buffer and acetonitrile in a volume ratio of 8:2, wherein the concentration of potassium hydrogen phosphate in the phosphate-chiral inclusion agent buffer was 90 mmol / L, the concentration of cyclodextrin was 80 mmol / L, and the pH of the phosphate-chiral inclusion agent buffer was adjusted to 3 with phosphoric acid; the mobile phase B was prepared by mixing acetonitrile and water in a volume ratio of 4:6; the detection wavelength was 215nm; the flow rate was 1.3mL / min; the column temperature was 30℃; the injection volume was 5μL; and the detector was an ultraviolet detector.

[0059] The elution gradient is: 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%; 2) When the elution time is 10 min, the volume fraction of mobile phase A is 100%; 3) When the elution time is 35 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; 4) When the elution time is 55 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; Test results such as Figure 6 ,from Figure 6 It can be seen that the separation between melogabalin (peak time is about 13 minutes) and various impurities meets the requirements.

[0060] Example 4 (1) Solution preparation: Same as Example 3 (2) Put the prepared solution into liquid chromatography for testing: The liquid chromatography conditions are as follows: except that the pH of the phosphate-chiral inclusion agent buffer is adjusted to 7.3 with phosphoric acid, the other conditions are the same as those in Example 1.

[0061] Test results such as Figure 7 ,from Figure 7 It can be seen that the separation between melogabalin (peak time is about 12 minutes) and various impurities meets the requirements.

[0062] Example 5 (1) Solution preparation: Same as Example 3 (2) Put the prepared solution into liquid chromatography for testing: The liquid chromatography conditions are as follows: except that the pH of the phosphate-chiral inclusion agent buffer is adjusted to 5.3 with phosphoric acid, the other conditions are the same as those in Example 1.

[0063] Test results such as Figure 8 ,from Figure 8 It can be seen that the separation between melogabalin (peak time is about 13 minutes) and various impurities meets the requirements.

[0064] 2. Methodological Validation Example 6 Method validation: After validation (results are shown in Table 1 below), all validations of the detection method described in the present invention can meet the standard requirements, indicating that this method can meet the requirements of the impurity spectrum study of melogabalin besylate raw material.

[0065] Table 1 Method validation results

[0066] Note: Melogabalin besylate will produce two peaks in the liquid chromatography system, namely benzenesulfonic acid peak and melogabalin peak. The melogabalin peak is the active ingredient peak of the drug. The area of ​​this peak is usually used as the quantitative parameter of the drug.

[0067] Example 7: Forced degradation test (1) Solution preparation: Blank solution: same as Example 1.

[0068] Test solution (acid degradation): Take about 40 mg of the test sample (melogabalin besylate raw material to be tested), place it in a 10 mL volumetric flask, add 1 mL of 1 mol / L HCl aqueous solution, place it at 60 ° C for 8 h, add 1 mol / L NaOH aqueous solution to adjust the pH to about 7 (determined using a wide range of pH test paper), add blank solvent to dilute to the scale, shake well, and use it as the acid degradation test solution.

[0069] (2) Put the prepared solution into liquid chromatography for testing: The test was carried out under the same liquid chromatography conditions as in Example 1.

[0070] Result analysis: Under acid degradation conditions, the separation between the melogabalin peak (peak time is about 13 minutes) and adjacent impurities and between known impurities can meet the separation requirements. The purity of the melogabalin peak is greater than 980 and the material conservation rate is between 95% and 105%, indicating that the method has good robustness and stability indication (see the specific results). Fig. 9 ).

[0071] Example 8: Forced degradation test (1) Solution preparation Blank solution: same as Example 1.

[0072] Test solution (alkaline degradation): Take about 40 mg of the test sample (melogabalin besylate raw material to be tested), place it in a 10 mL volumetric flask, add 1 mL of 1 mol / L NaOH aqueous solution, place it at 60 ° C for 8 h, add 1 mol / L HCl aqueous solution to adjust the pH to about 7 (determined using a wide range of pH test paper), add blank solvent to dilute to the scale, shake well, and use it as the alkaline degradation test solution.

[0073] (2) Put the prepared solution into liquid chromatography for testing: The test was carried out under the same liquid chromatography conditions as in Example 1.

[0074] Result analysis: Under alkaline degradation conditions, the separation between the melogabalin peak (peak time is about 12 minutes) and adjacent impurities and between known impurities can meet the separation requirements. The purity of the melogabalin peak is greater than 980 and the material conservation rate is between 95% and 105%, indicating that the method has good robustness and stability indication (see the specific results). Fig.10 ).

[0075] Example 9: Forced degradation test (1) Solution preparation Blank solution: same as Example 1.

[0076] Test solution (oxidative degradation): Take about 40 mg of the test sample (merogabalin besylate raw material to be tested) and place it in a 10 ml volumetric flask. Add 1 ml of 3% H 2 O 2 The solution was placed at 60 °C for 8 h, diluted to the scale with blank solvent, and shaken well to serve as the oxidative degradation test solution.

[0077] (2) Put the prepared solution into liquid chromatography for testing: The test was carried out under the same liquid chromatography conditions as in Example 1.

[0078] Result analysis: Under the conditions of oxidative degradation, the separation between the melogabalin peak (peak time is about 12 minutes) and the adjacent impurities and between the known impurities can meet the separation requirements. The purity of the melogabalin peak is greater than 980 and the material conservation rate is between 95% and 105%, indicating that the method has good robustness and stability indication (see the specific results). Fig.11 ).

[0079] Comparative Example 1: (Refer to original patent CN104755456A) (1) Solution preparation: Blank solution: same as Example 1.

[0080] Preparation of test solution: Take an appropriate amount of the product (melogabalin besylate raw material to be tested), accurately weigh it, add blank solvent to dissolve and dilute it to make a solution containing 5.0 mg of the product per 1 mL.

[0081] (2) Liquid chromatography conditions are: Chromatographic column: Cadenza CW-C18 (I imtakt, 4.6×150mm, 3μm); 5mmol / L ammonium bicarbonate aqueous solution as mobile phase A, acetonitrile as mobile phase B; detection wavelength: 205nm; flow rate: 1.0mL / min; column temperature: 40℃; injection volume: 2μL; elution time: 60min; detector: UV detector.

[0082] The elution gradient is: 1) At elution time 0 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%; 2) When the elution time is 12 min, the volume fraction of mobile phase A is 90% and the volume fraction of mobile phase B is 10%; 3) When the elution time is 27 min, the volume fraction of mobile phase A is 20%, and the volume fraction of mobile phase B is 80%; 4) When the elution time is 45 min, the volume fraction of mobile phase A is 20% and the volume fraction of mobile phase B is 80%; 5) When the elution time is 50 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%; 6) When the elution time is 60 min, the volume fraction of mobile phase A is 90% and the volume fraction of mobile phase B is 10%.

[0083] Conclusion analysis: This method uses conventional reverse phase chromatography, and no cyclodextrin is added to the mobile phase. The peak of melogabalin cannot be separated from the peak of diastereoisomer impurity (impurity 1), and impurity 1 and impurity 2 cannot be separated at all. It cannot meet the research requirements of melogabalin besylate raw material impurities (for specific results, see Fig.12 ).

[0084] Comparative Example 2: (Mobile phase system without cyclodextrin) (1) Solution preparation: Blank solvent: acetonitrile and water were mixed evenly in a volume ratio of 1:9.

[0085] Test solution: Take an appropriate amount of this product (melogabalin besylate API to be tested), dissolve it in blank solvent and dilute it to make a solution containing approximately 2.5 mg of melogabalin besylate API to be tested per 1 mL.

[0086] System suitability solution: Take appropriate amount of this product (melogabalin besylate API to be tested), impurity 1 reference substance, impurity 2 reference substance, impurity 3 reference substance and impurity 4 reference substance, add appropriate amount of blank solvent to dissolve, and shake well to obtain.

[0087] (2) Accurately measure the test solution and system suitability solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0088] The liquid chromatography conditions were: Octylsilane bonded silica gel was used as filler (Eclipse XDB C8, 4.6×150mm, 3.5μm), and dipotassium hydrogen phosphate solution and acetonitrile were mixed in a volume ratio of 9:1 to obtain mobile phase A, the concentration of dipotassium hydrogen phosphate solution was 10mmol / L, and the pH of the dipotassium hydrogen phosphate solution was adjusted to 6.5 with phosphoric acid; acetonitrile was used as mobile phase B, and linear gradient elution was performed; the detection wavelength was 205nm; the column temperature was 40℃; the flow rate was 1.0mL per minute; and the injection volume was 5μL.

[0089] The elution gradient is: 1) At elution time 0 min, the volume fraction of mobile phase A is 96%, and the volume fraction of mobile phase B is 4%; 2) When the elution time is 20 min, the volume fraction of mobile phase A is 96%, and the volume fraction of mobile phase B is 4%; 3) When the elution time is 30 min, the volume fraction of mobile phase A is 30% and the volume fraction of mobile phase B is 70%; 4) When the elution time is 35 min, the volume fraction of mobile phase A is 30% and the volume fraction of mobile phase B is 70%; 5) When the elution time is 35.1 min, the volume fraction of mobile phase A is 96%, and the volume fraction of mobile phase B is 4%; 6) When the elution time is 60 min, the volume fraction of mobile phase A is 96% and the volume fraction of mobile phase B is 4%.

[0090] Conclusion Analysis: This method uses conventional reverse phase chromatography, no cyclodextrin is added to the mobile phase, and the content of diastereoisomer impurity (impurity 1) is high (equivalent to 1% of the concentration of the test solution, see attached Fig.13 ), the separation degree between the melogabalin benzenesulfonate peak and it can be displayed as 2.1, but when the impurity 1 content is less than 0.1%, the separation degree between the two cannot be effectively displayed (see Appendix Fig.14 ), and thus cannot meet the application requirements; in addition, under the conditions of this method, impurity 1 and impurity 2 cannot be separated at all, and it is not suitable for the analysis and research of impurities in melogabalin besylate raw material.

[0091] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are exhaustively listed. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0092] For those skilled in the art, several modifications and improvements may be made without departing from the concept of the present invention, all of which belong to the protection scope of the present invention. The protection scope of the present invention shall be based on the attached claims.

Claims

1. A method for analyzing the impurity spectrum of melogabalin besylate raw material drug, characterized in that: The analysis method is: S1. Preparation of solution: Preparation of blank solvent: Mix acetonitrile and water evenly to obtain blank solvent; Preparation of test solution: Accurately weigh the melogabalin besylate bulk drug substance to be tested, dissolve it in blank solvent and dilute it to make a test solution containing 4.0 mg per 1 ml; Preparation of control solution: accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add blank solvent to dilute to the scale, and shake well; accurately measure 1 mL of the solution in the 100 mL volumetric flask, place it in a 10 mL volumetric flask, add blank solvent to dilute to the scale, and shake well to obtain the control solution; S2. Use liquid chromatography to detect the test solution and the control solution: The detector of the liquid chromatography is an ultraviolet detector, and the chromatographic conditions are: mobile phase A includes phosphate-chiral inclusion agent buffer, mobile phase B includes acetonitrile and water, and gradient elution is adopted; the flow rate is 0.5-2.0 mL / min; the chromatographic column stationary phase filler is non-chiral silane bonded silica gel; the column temperature is 15°C-55°C; and the detection wavelength is 190nm-230nm.

2. A method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 1, characterized in that: The chiral inclusion agent is at least one of cyclodextrin, crown ether, vancomycin and norvancomycin.

3. A method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 1, characterized in that: The pH value of the mobile phase A is 2.0-8.8; the phosphate concentration in the phosphate-chiral inclusion agent buffer of the mobile phase A is 1-200 mmol / L; the chiral inclusion agent concentration in the phosphate-chiral inclusion agent buffer is 1-200 mmol / L; The mobile phase A contains acetonitrile or does not contain acetonitrile, and the volume ratio of the phosphate-chiral inclusion agent buffer to acetonitrile in the mobile phase A is (6:4) to (10:0).

4. A method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 3, characterized in that: The pH value of the mobile phase A is 3.0-4.0; the phosphate concentration in the phosphate-chiral inclusion agent buffer of the mobile phase A is 70-90 mmol / L; the chiral inclusion agent concentration in the phosphate-chiral inclusion agent buffer is 80-120 mmol / L; The volume ratio of the phosphate-chiral inclusion agent buffer to acetonitrile in the mobile phase A is (8:2) to (9.5:0.5).

5. The method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 1, characterized in that: The mobile phase B contains methanol or does not contain methanol, and the volume ratio of acetonitrile, water and methanol in the mobile phase B is (4:6:0) to (8:1:1).

6. A method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 5, characterized in that: The volume ratio of acetonitrile, water and methanol in the mobile phase B is 6:4:0.

5.

7. A method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 1, characterized in that: The non-chiral alkyl group bonded to the silica gel in the chromatographic column stationary phase filler is selected from any one of octadecyl, octyl, phenyl, phenylhexyl, pentafluorophenyl and octadecyl-pentafluorophenyl.

8. The method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 1, characterized in that: The flow rate is 0.8-1.5 mL / min; the column temperature is 20° C.-30° C.; and the detection wavelength is 205 nm-215 nm.

9. The method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 1, characterized in that: The gradient elution is: 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%; 2) When the elution time is 10 min, the volume fraction of mobile phase A is 100%; 3) When the elution time is 35 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%; 4) When the elution time is 55 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%.

10. A method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 1, characterized in that: In the blank solvent, the volume ratio of acetonitrile to water is 2:8.

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