An analytical method for the impurity profile of pregabalin mesylate bulk drug

Abstract

The present invention relates to the technical field of pharmaceutical detection, and particularly relates to an analytical method for the impurity profile of megalol gabapentin besylate raw material drugs. The method uses high performance liquid chromatography with a chiral complexing agent added to the mobile phase for determination. The liquid chromatography conditions are as follows: mobile phase A includes a phosphate-chiral complexing agent buffer solution, 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 achiral silane-bonded silica gel; the column temperature is 15°C - 55°C; the detection wavelength is 190 nm - 230 nm. This method has the advantages of simple operation, low cost, good selectivity, high accuracy, and strong universality. It is suitable for the study of the impurity profile of megalol gabapentin besylate raw material drugs, thereby effectively controlling the product quality. At the same time, it can be extended to the study of the impurity profiles of other amino acids and their similar drugs.

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] .

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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

[0012] 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.

[0013] The technical solution for the present invention to solve the above technical problems is as follows: An analytical method for the impurity profile of megalolabaline besylate bulk drug, and the analytical method is as follows:

[0014] S1. Preparation of solutions:

[0015] Preparation of blank solvent: Mix acetonitrile and water evenly to obtain the blank solvent;

[0016] Preparation of test solution: Weigh accurately the megalolabaline besylate bulk drug to be tested, dissolve and dilute it with the blank solvent to prepare a test solution containing 4.0 mg per 1 mL;

[0017] Preparation of control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute it to the mark with the blank solvent, and shake well; then accurately measure 1 mL of the solution in the 100 mL volumetric flask, place it in a 10 mL volumetric flask, dilute it to the mark with the blank solvent, and shake well to obtain the control solution;

[0018] S2. Detect the test solution and the control solution using liquid chromatography:

[0019] The detector of the liquid chromatography is an ultraviolet detector, and the chromatographic conditions are as follows: Mobile phase A includes phosphate-chiral complexing agent buffer solution, mobile phase B includes acetonitrile and water, and gradient elution is adopted; the flow rate is 0.5 - 2.0 mL / min; the stationary phase packing material of the chromatographic column is achiral silane-bonded silica gel; the column temperature is 15°C - 55°C; the detection wavelength is 190 nm - 230 nm.

[0020] Furthermore, the chiral complexing agent is at least one of cyclodextrin, crown ether, vancomycin, and norvancomycin.

[0021] Furthermore, the pH value of mobile phase A is 2.0 - 8.8; the phosphate concentration in the phosphate-chiral complexing agent buffer solution of mobile phase A is 1 - 200 mmol / L; the chiral complexing agent concentration in the phosphate-chiral complexing agent buffer solution is 1 - 200 mmol / L;

[0022] Mobile phase A contains acetonitrile or does not contain acetonitrile, and the volume ratio of the phosphate-chiral complexing agent buffer solution to acetonitrile in mobile phase A is (6:4) - (10:0).

[0023] Preferably, the pH value of mobile phase A is 3.0 - 4.0; the phosphate concentration in the phosphate-chiral complexing agent buffer solution of mobile phase A is 70 - 90 mmol / L; the chiral complexing agent concentration in the phosphate-chiral complexing agent buffer solution is 80 - 120 mmol / L;

[0024] The volume ratio of the phosphate-chiral inclusion complex buffer solution to acetonitrile in the mobile phase A is (8:2) to (9.5:0.5).

[0025] 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).

[0026] Preferably, the volume ratio of acetonitrile, water, and methanol in the mobile phase B is 6:4:0.5.

[0027] Furthermore, the achiral alkyl group bonded to silica gel in the chromatographic column stationary phase filler is selected from any one of octadecyl, octyl, phenyl, phenylhexyl, pentafluorophenyl, and octadecyl-pentafluorophenyl.

[0028] Preferably, the flow rate is 0.8 - 1.5 mL / min; the column temperature is 20°C - 30°C; the detection wavelength is 205 nm - 215 nm.

[0029] Furthermore, the gradient elution is as follows:

[0030] 1) At an elution time of 0 min, the volume fraction of the mobile phase A is 100%;

[0031] 2) At an elution time of 10 min, the volume fraction of the mobile phase A is 100%;

[0032] 3) At an elution time of 35 min, the volume fraction of the mobile phase A is 50%, and the volume fraction of the mobile phase B is 50%;

[0033] 4) At an elution time of 55 min, the volume fraction of the mobile phase A is 50%, and the volume fraction of the mobile phase B is 50%.

[0034] Furthermore, in the blank solvent, the volume ratio of acetonitrile to water is 2:8.

[0035] The beneficial effects of the present invention are:

[0036] 1. The present invention uses the chiral inclusion complex method for a comprehensive study of the impurity profile of megalolabalin benzenesulfonate, which not only involves chiral isomer impurities, but also includes residual raw materials, achiral isomer impurities, genotoxic impurities, degradation impurities, and other process by-products.

[0037] 2. By adding an inexpensive chiral complexing agent, such as cyclodextrin, to the mobile phase, and utilizing the inclusion effect of the internal cavity structure of cyclodextrin on isomers to form chiral inclusion compounds with different physicochemical properties, different partition coefficients are generated in the mobile phase and the stationary phase, effectively achieving complete baseline separation of various types of isomeric impurities (including enantiomeric impurities). Even trace impurity residues can meet the resolution requirements. At the same time, the use of expensive chiral chromatographic columns is avoided, solving the problems of "difficult separation of isomeric impurities, difficult quantification of trace components, and poor regulatory compliance", significantly reducing the testing cost, and also providing a technical basis for the separation of isomeric impurities of other compounds.

[0038] 3. Melogabalin benzenesulfonate has weak ultraviolet absorption and is a terminal absorption. The choice of additives has strong limitations. Cyclodextrin has a low ultraviolet absorption cut-off wavelength and basically no ultraviolet absorption, and has the advantages of rapid and stable inclusion, does not interfere with the determination of each target impurity, and has good repeatability and accuracy of results.

[0039] 4. The present invention uses a conventional detector to effectively monitor the genotoxic impurity ethyl benzenesulfonate in melogabalin benzenesulfonate raw material (the detection limit can reach 10 ppm), avoiding the use of expensive mass spectrometric detectors, reducing the detection cost, and expanding the universality of this method.

[0040] 5. The present invention adopts high performance liquid chromatography, which requires no complex pretreatment, has low cost, strong universality, high precision, good reproducibility, has a strong stability indicating effect, and can be smoothly transferred to different laboratories for quality control of melogabalin benzenesulfonate raw material, thereby ensuring its safety, effectiveness and quality controllability. Description of the Drawings

[0041] Figure 1 It is the chromatogram of the blank solution in Example 1;

[0042] Figure 2 It is the chromatogram of the mixed solution in Example 1;

[0043] Figure 3 It is the chromatogram of the test solution in Example 1;

[0044] Figure 4 It is the chromatogram of the control solution in Example 1;

[0045] Figure 5 It is the chromatogram of the mixed solution in Example 2;

[0046] Figure 6 It is the chromatogram of the spiked test solution in Example 3;

[0047] Figure 7 It is the chromatogram of the spiked test solution in Example 4;

[0048] Figure 8 It is the chromatogram of the spiked test sample solution in Example 5;

[0049] Figure 9 It is the chromatogram of the test sample solution (acid degradation) in Example 7;

[0050] Figure 10 It is the chromatogram of the test sample solution (base degradation) in Example 8;

[0051] Figure 11 It is the chromatogram of the test sample solution (oxidative degradation) in Example 9;

[0052] Figure 12 It is the chromatogram of the test sample solution in Comparative Example 1;

[0053] Figure 13 It is the chromatogram of the system suitability solution in Comparative Example 2;

[0054] Figure 14 It is the chromatogram of the test sample solution in Comparative Example 2. Detailed implementation manners

[0055] The following makes a detailed description of the specific implementation manners of the present invention. The present invention can be implemented in many other ways different from those described herein. Those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the disclosed specific embodiments.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used are only for describing the specific implementation manners and do not limit the present invention.

[0057] An analytical method for the impurity profile of megalol gabapentin benzenesulfonate raw material drug, the analytical method is as follows:

[0058] S1. Preparation of solutions:

[0059] Preparation of the blank solvent: The blank solvent is obtained by mixing acetonitrile and water evenly;

[0060] Preparation of the test sample solution: Weigh precisely the megalol gabapentin benzenesulfonate raw material drug to be tested, dissolve and dilute it with the blank solvent to prepare a test sample solution containing 4.0 mg per 1 mL;

[0061] Preparation of the control solution: Precisely measure 1 mL of the test sample solution and place it in a 100-mL volumetric flask, dilute it to the mark with the blank solvent, and shake well; then precisely measure 1 mL of the solution in the 100-mL volumetric flask and place it in a 10-mL volumetric flask, dilute it to the mark with the blank solvent, and shake well to obtain the control solution;

[0062] S2. Detect the test solution and the reference solution using liquid chromatography:

[0063] The detector of the liquid chromatography is an ultraviolet detector, and the chromatographic conditions are as follows: Mobile phase A contains a phosphate-chiral complexing agent buffer solution, mobile phase B contains acetonitrile and water, and gradient elution is adopted; the flow rate is 0.5 - 2.0 mL / min; the stationary phase filler of the chromatographic column is achiral silane-bonded silica gel; the column temperature is 15°C - 55°C; the detection wavelength is 190 nm - 230 nm.

[0064] Specifically, the chiral complexing agent is at least one of cyclodextrin, crown ether, vancomycin, and norvancomycin.

[0065] Specifically, the pH value of mobile phase A is 2.0 - 8.8; the phosphate concentration in the phosphate-chiral complexing agent buffer solution of mobile phase A is 1 - 200 mmol / L; the chiral complexing agent concentration in the phosphate-chiral complexing agent buffer solution is 1 - 200 mmol / L;

[0066] Mobile phase A contains acetonitrile or does not contain acetonitrile, and the volume ratio of the phosphate-chiral complexing agent buffer solution to acetonitrile in mobile phase A is (6:4) - (10:0).

[0067] Preferably, the pH value of mobile phase A is 3.0 - 4.0; the phosphate concentration in the phosphate-chiral complexing agent buffer solution of mobile phase A is 70 - 90 mmol / L; the chiral complexing agent concentration in the phosphate-chiral complexing agent buffer solution is 80 - 120 mmol / L;

[0068] The volume ratio of the phosphate-chiral complexing agent buffer solution to acetonitrile in mobile phase A is (8:2) - (9.5:0.5).

[0069] Among them, the phosphate-chiral complexing agent buffer solution is an aqueous solution prepared with phosphate and a chiral complexing agent, and is adjusted to an appropriate pH with phosphoric acid.

[0070] Specifically, mobile phase B contains methanol or does not contain methanol, and the volume ratio of acetonitrile, water, and methanol in mobile phase B is (4:6:0) - (8:1:1).

[0071] Preferably, the volume ratio of acetonitrile, water, and methanol in mobile phase B is 6:4:0.5.

[0072] Specifically, the achiral alkyl group bonded to silica gel in the stationary phase filler of the chromatographic column is selected from any one of octadecyl, octyl, phenyl, phenylhexyl, pentafluorophenyl, and octadecyl-pentafluorophenyl.

[0073] Preferably, the achiral alkyl group bonded to silica in the chromatographic column stationary phase filler is phenyl.

[0074] Preferably, the flow rate is 0.8 - 1.5 mL / min; the column temperature is 20°C - 30°C; the detection wavelength is 205 nm - 215 nm.

[0075] Specifically, the gradient elution is as follows:

[0076] 1) At an elution time of 0 min, the volume fraction of mobile phase A is 100%;

[0077] 2) At an elution time of 10 min, the volume fraction of mobile phase A is 100%;

[0078] 3) At an elution time of 35 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%;

[0079] 4) At an elution time of 55 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%.

[0080] Specifically, in the blank solvent, the volume ratio of acetonitrile to water is 2:8.

[0081] More specifically, the chromatographic column is selected from any one of ZORBAX Phenyl, Eclipse Plus Phenyl - Hexyl, and ACE Excel3 C18 - PFP.

[0082] During the test, accurately measure the control solution and the test solution in step S1, inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate the impurity content according to the self - control method of melogabalin, and the calculation formula is:

[0083] Impurity content (%) = As / Ar × 0.1%

[0084] In the formula:

[0085] As—the peak area of each impurity in the test solution;

[0086] Ar—the peak area of the main peak in the control solution.

[0087] I. Detection Examples

[0088] Example 1

[0089] (1) Solution preparation:

[0090] Blank solvent: A solution prepared from acetonitrile and water (the volume ratio of acetonitrile to water is 2:8).

[0091] 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.

[0092] 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.

[0093] 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.

[0094] (2) Put the prepared solution into liquid chromatography for testing:

[0095] The liquid chromatography conditions were:

[0096] The chromatographic column was ZORBAX Phenyl (4.6×150mm, 5μm); the mobile phase A was prepared by the volume ratio of phosphate-chiral inclusion agent buffer and acetonitrile 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 the volume ratio of acetonitrile, water and methanol 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; the detector was an ultraviolet detector.

[0097] The elution gradient is:

[0098] 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%;

[0099] 2) When the elution time is 10 min, the volume fraction of mobile phase A is 100%;

[0100] 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%;

[0101] 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%;

[0102] Test results such as Figures 1-4 ,from Figures 1-4It can be seen that the resolution between melegabalin (the peak emergence time is about 13 minutes. Melegabalin besylate will show two peaks in the liquid chromatography system, namely the peak of besylate and the peak of melegabalin. Among them, the peak of melegabalin is the active ingredient peak of the drug, and usually the area of this peak is used as the quantitative parameter of the drug) and each impurity meets the requirements. The resolution between impurity 1 and impurity 2 can meet the separation requirements, and the resolution between other known impurities and known impurities can also meet the separation requirements.

[0103] Example 2

[0104] (1) Solution preparation:

[0105] Blank solvent: A solution prepared from acetonitrile and water (the volume ratio of acetonitrile to water is 2:8).

[0106] Mixed solution: Appropriate amounts of melegabalin 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 were taken, dissolved with an appropriate amount of solvent, and 1 mL was taken and transferred to a 100 mL volumetric flask, diluted to the mark with the solvent, and shaken well to obtain.

[0107] (2) Inject the prepared solution into the liquid chromatography for testing:

[0108] The liquid chromatography conditions are as follows:

[0109] The chromatographic column is Eclipse Plus Phenyl-Hexyl (4.6×150 mm, 4 μm); Mobile phase A is prepared by mixing phosphate-chiral complexing agent buffer solution and acetonitrile according to a volume ratio of 9.5:0.5, where the concentration of dipotassium hydrogen phosphate in the phosphate-chiral complexing agent buffer solution is 70 mmol / L, the concentration of crown ether is 120 mmol / L, and the pH of the phosphate-chiral complexing agent buffer solution is adjusted to 4 with phosphoric acid; Mobile phase B is prepared by mixing acetonitrile, water, and methanol according to a volume ratio of 8:1:1; The detection wavelength is 205 nm; The flow rate is 0.8 mL / min; Column temperature: 20 °C; The injection volume is 5 μL; The detector is an ultraviolet detector.

[0110] The elution gradient is as follows:

[0111] 1) At the elution time of 0 min, the volume fraction of mobile phase A is 100%;

[0112] 2) At the elution time of 10 min, the volume fraction of mobile phase A is 100%;

[0113] 3) At the elution time of 35 min, the volume fraction of mobile phase A is 50%, and the volume fraction of mobile phase B is 50%;

[0114] 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%.

[0115] The detection results are as Figure 5 , from Figure 5 it can be seen that the resolution between melegabalin (peak elution time is about 12 minutes) and each impurity meets the requirements.

[0116] Example 3

[0117] (1) Solution preparation:

[0118] Blank solvent: A solution prepared from acetonitrile and water (volume ratio of acetonitrile to water is 2:8).

[0119] Preparation of test solution: Take an appropriate amount of this product (melegabalin besylate raw material to be tested), weigh accurately, dissolve and dilute with the blank solvent to prepare a solution containing 4.0 mg of this product per 1 mL.

[0120] Preparation of control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the mark with the blank solvent, and shake well; accurately measure 1 mL of the above solution, place it in a 10 mL volumetric flask, dilute to the mark with the blank solvent, and shake well to obtain the control solution.

[0121] Spiked test solution: Take appropriate amounts of this product (melegabalin besylate raw material to be tested), reference substance of impurity 1, reference substance of impurity 2, reference substance of impurity 3, reference substance of impurity 4, reference substance of impurity 5, reference substance of impurity 6, and reference substance of impurity 7, dissolve with an appropriate amount of solvent, and take 1 mL into a 100 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain.

[0122] (2) Inject the prepared solutions into the liquid chromatograph for testing:

[0123] The liquid chromatography conditions are as follows:

[0124] Chromatographic column: ACE Excel 3 C18-PFP (4.6×150 mm, 3 μm); Phosphate-chiral complexing agent buffer solution and acetonitrile are prepared into mobile phase A according to a volume ratio of 8:2, where the concentration of dipotassium hydrogen phosphate in the phosphate-chiral complexing agent buffer solution is 90 mmol / L, the concentration of cyclodextrin is 80 mmol / L, and the pH of the phosphate-chiral complexing agent buffer solution is adjusted to 3 with phosphoric acid; Mobile phase B is prepared from acetonitrile and water according to a volume ratio of 4:6; Detection wavelength: 215 nm; Flow rate: 1.3 mL / min; Column temperature: 30 °C; Injection volume: 5 μL; Detector: Ultraviolet detector.

[0125] The elution gradient is as follows:

[0126] 1) At an elution time of 0 min, the volume fraction of mobile phase A is 100%;

[0127] 2) At an elution time of 10 min, the volume fraction of mobile phase A is 100%;

[0128] 3) At an elution time of 35 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%;

[0129] 4) At an elution time of 55 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%;

[0130] The test results are as Figure 6 , from Figure 6 it can be seen that the resolution between melegabalin (peak elution time is about 13 minutes) and each impurity meets the requirements.

[0131] Example 4

[0132] (1) Solution preparation: The same as in Example 3

[0133] (2) Inject the prepared solution into the liquid chromatograph for testing:

[0134] The liquid chromatographic conditions are as follows: Except that the pH of the phosphate - chiral complexing agent buffer solution is adjusted to 7.3 with phosphoric acid, the other conditions are the same as in Example 1.

[0135] The test results are as Figure 7 , from Figure 7 it can be seen that the resolution between melegabalin (peak elution time is about 12 minutes) and each impurity meets the requirements.

[0136] Example 5

[0137] (1) Solution preparation: The same as in Example 3

[0138] (2) Inject the prepared solution into the liquid chromatograph for testing:

[0139] The liquid chromatographic conditions are as follows: Except that the pH of the phosphate - chiral complexing agent buffer solution is adjusted to 5.3 with phosphoric acid, the other conditions are the same as in Example 1.

[0140] The test results are as Figure 8 , from Figure 8 it can be seen that the resolution between melegabalin (peak elution time is about 13 minutes) and each impurity meets the requirements.

[0141] II. Verification of Methodology

[0142] Example 6

[0143] Method Validation: After verification (the 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 for the impurity profile study of melegabalin besylate raw materials.

[0144] Table 1 Method Validation Results

[0145]

[0146] Note: Melegabalin besylate will show two peaks in the liquid chromatography system, namely the peak of benzenesulfonic acid and the peak of melegabalin. Among them, the peak of melegabalin is the active ingredient peak of the drug, and usually the area of this peak is used as the quantitative parameter of the drug.

[0147] Example 7: Forced Degradation Test

[0148] (1) Solution Preparation:

[0149] Blank Solution: The same as in Example 1.

[0150] Test Solution (Acid Degradation): Take about 40 mg of the test substance (melegabalin 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 (measured using a wide-range pH test paper), dilute to the mark with blank solvent, and shake well to obtain the acid degradation test solution.

[0151] (2) Inject the prepared solutions into the liquid chromatography for testing:

[0152] Use the same liquid chromatography conditions as in Example 1 for testing.

[0153] Result Analysis: Under the acid degradation conditions, the resolution between the melegabalin peak (retention time is about 13 minutes) and adjacent impurities, and between each known impurity can reach the separation requirements. The purity of the melegabalin peak is greater than 980 and the material balance rate is between 95% and 105%, indicating that this method has good robustness and stability indicating effect (the specific results are shown in Figure 9 )

[0154] Example 8: Forced Degradation Test

[0155] (1) Solution Preparation

[0156] Blank Solution: The same as in Example 1.

[0157] Test solution (alkaline degradation): Take about 40 mg of the test substance (melogabalin benzenesulfonate API to be determined), place it in a 10 mL volumetric flask, add 1 mL of 1 mol / L NaOH aqueous solution, let it stand at 60 °C for 8 h, add 1 mol / L HCl aqueous solution to adjust the pH to about 7 (measured using a wide-range pH test paper), dilute to the mark with blank solvent, shake well, and use it as the test solution for alkaline degradation.

[0158] (2) Inject the prepared solution into the liquid chromatograph for testing:

[0159] Conduct the test under the same liquid chromatography conditions as in Example 1.

[0160] Result analysis: Under the alkaline degradation conditions, the resolution between the melogabalin peak (retention time is about 12 minutes) and adjacent impurities, as well as between each known impurity, can meet the separation requirements. The purity of the melogabalin peak is greater than 980 and the material balance rate is between 95% and 105%, indicating that this method has good robustness and stability indicating effect (for specific results, see Figure 10 )

[0161] Example 9: Forced degradation test

[0162] (1) Solution preparation

[0163] Blank solution: The same as in Example 1.

[0164] Test solution (oxidative degradation): Take about 40 mg of the test substance (melogabalin benzenesulfonate API to be determined), place it in a 10 mL volumetric flask, add 1 mL of 3% H2O2 solution, let it stand at 60 °C for 8 h, dilute to the mark with blank solvent, shake well, and use it as the test solution for oxidative degradation.

[0165] (2) Inject the prepared solution into the liquid chromatograph for testing:

[0166] Conduct the test under the same liquid chromatography conditions as in Example 1.

[0167] Result analysis: Under the oxidative degradation conditions, the resolution between the melogabalin peak (retention time is about 12 minutes) and adjacent impurities, as well as between each known impurity, can meet the separation requirements. The purity of the melogabalin peak is greater than 980 and the material balance rate is between 95% and 105%, indicating that this method has good robustness and stability indicating effect (for specific results, see Figure 11 )

[0168] Comparative Example 1: (Refer to the original research patent CN104755456A)

[0169] (1) Solution preparation:

[0170] Blank solution: same as in Example 1.

[0171] Preparation of test solution: Take an appropriate amount of this product (the raw material of megalolabalin benzenesulfonate to be tested), weigh accurately, dissolve and dilute with the blank solvent to prepare a solution containing 5.0 mg of this product per 1 mL.

[0172] (2) The liquid chromatography conditions are as follows:

[0173] Chromatographic column: Cadenza CW-C18 (I imtakt, 4.6×150 mm, 3 μm); 5 mmol / L ammonium bicarbonate aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B; the detection wavelength is 205 nm; the flow rate is 1.0 mL / min; the column temperature is 40 °C; the injection volume is 2 μL; the elution time is 60 min; the detector is an ultraviolet detector.

[0174] The elution gradient is as follows:

[0175] 1) At the elution time of 0 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%;

[0176] 2) At the elution time of 12 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%;

[0177] 3) At the elution time of 27 min, the volume fraction of mobile phase A is 20%, and the volume fraction of mobile phase B is 80%;

[0178] 4) At the elution time of 45 min, the volume fraction of mobile phase A is 20%, and the volume fraction of mobile phase B is 80%;

[0179] 5) At the elution time of 50 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%;

[0180] 6) At the elution time of 60 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%.

[0181] Conclusion analysis: Under this method, the conventional reverse-phase chromatography method is adopted, and cyclodextrin is not added to the mobile phase. The peak of megalolabalin and the peak of diastereoisomeric impurity (impurity 1) cannot be separated, and impurity 1 and impurity 2 cannot be separated completely, which cannot meet the requirements for impurity research of the raw material of megalolabalin benzenesulfonate (for specific results, see Figure 12 )

[0182] Comparative Example 2: (Mobile phase system without cyclodextrin)

[0183] (1) Solution preparation:

[0184] Blank solvent: Prepared by mixing acetonitrile and water in a volume ratio of 1:9 evenly.

[0185] Test solution: Take an appropriate amount of this product (the raw material drug of melegabalin benzenesulfonate to be tested), dissolve and dilute it with the blank solvent to prepare a solution containing about 2.5 mg of the raw material drug of melegabalin benzenesulfonate to be tested per 1 mL.

[0186] System suitability solution: Take appropriate amounts of this product (the raw material drug of melegabalin benzenesulfonate to be tested), reference substance for impurity 1, reference substance for impurity 2, reference substance for impurity 3, and reference substance for impurity 4 respectively, dissolve them with an appropriate amount of the blank solvent, and shake well to obtain.

[0187] (2) Precisely measure the test solution and the system suitability solution, and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0188] The liquid chromatography conditions are as follows:

[0189] Use octadecylsilane-bonded silica gel as the filler (Eclipse XDB C8, 4.6×150 mm, 3.5 μm), mix potassium hydrogen phosphate solution and acetonitrile evenly according to the volume ratio of 9:1 to obtain mobile phase A, the concentration of potassium hydrogen phosphate solution is 10 mmol / L, and the pH of potassium hydrogen phosphate solution is adjusted to 6.5 with phosphoric acid; acetonitrile is mobile phase B, and linear gradient elution is carried out; the detection wavelength is 205 nm; the column temperature is 40 °C; the flow rate is 1.0 mL per minute; the injection volume is 5 μL.

[0190] The elution gradient is as follows:

[0191] 1) At the elution time of 0 min, the volume fraction of mobile phase A is 96%, and the volume fraction of mobile phase B is 4%;

[0192] 2) At the elution time of 20 min, the volume fraction of mobile phase A is 96%, and the volume fraction of mobile phase B is 4%;

[0193] 3) At the elution time of 30 min, the volume fraction of mobile phase A is 30%, and the volume fraction of mobile phase B is 70%;

[0194] 4) At the elution time of 35 min, the volume fraction of mobile phase A is 30%, and the volume fraction of mobile phase B is 70%;

[0195] 5) At the elution time of 35.1 min, the volume fraction of mobile phase A is 96%, and the volume fraction of mobile phase B is 4%;

[0196] 6) At the elution time of 60 min, the volume fraction of mobile phase A is 96%, and the volume fraction of mobile phase B is 4%.

[0197] Conclusion analysis: Under this method, the conventional reverse-phase chromatography was adopted, and cyclodextrin was not added to the mobile phase. When the content of the diastereomeric impurity (Impurity 1) was relatively high (equivalent to the 1% level of the test solution concentration, see Appendix Figure 13 ), the resolution between the meloxicam peak and it could be shown as 2.1. However, when the content of Impurity 1 was less than 0.1%, the resolution between the two could not be effectively shown (see Appendix Figure 14 ), thus unable to meet the declaration requirements. In addition, under the conditions of this method, Impurity 1 and Impurity 2 could not be separated at all, and it was not applicable to the analysis and research of the impurities in meloxicam raw materials.

[0198] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, all possible combinations of the various technical features in the above embodiments are not exhaustively listed. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0199] For those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. The protection scope of the present invention shall be subject to the appended 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: the mobile phase A includes a phosphate-chiral inclusion agent buffer, the mobile phase B includes acetonitrile and water, and a gradient elution method 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.; the detection wavelength is 190 nm-230 nm; The chiral inclusion agent is at least one of cyclodextrin, crown ether, vancomycin, and norvancomycin; 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.

2. 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).

3. A method for analyzing the impurity spectrum of melogabalin besylate bulk drug according to claim 2, 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).

4. A 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).

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

5.

6. A 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.

7. 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%.

8. The 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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