Quality control method of melogabalin besylate

Abstract

The invention discloses a quality control method of melogabalin besylate, which can effectively elute, separate and quantify various specific impurities in melogabalin besylate by optimizing chromatographic conditions and adopting gradient elution, and has the advantages of strong specificity, wide linear range, high sensitivity, good accuracy and good solution stability and durability. The quality control of the melogabalin besylate raw material medicine and the preparation product thereof is facilitated.

Description

Technical Field

[0001] The invention relates to a quality control method for melogabalin besylate, in particular to a quality control method for impurities in melogabalin besylate. Background Art

[0002] Melegabalin besylate, as a GABA analog, is a new type of preferentially selective α2δ-1 ligand that acts on the α2-δ1 subunit of the voltage-sensitive calcium channel complex and is used to treat pain associated with diabetic peripheral neuropathy, fibromyalgia and postherpetic neuralgia.

[0003]

[0004] The synthesis route of melogabalin besylate is as follows. The synthesis process will produce 5 specific impurities, which can be transferred to the raw materials and even remain in the finished products through the subsequent synthesis process, which will have an adverse effect on the quality of the raw materials and finished products. However, there is currently no effective detection method for these specific impurities.

[0005] Summary of the invention

[0006] Purpose of the invention: The present invention aims to provide a quality control method capable of effectively eluting, separating and quantifying specific impurities in milogabalin besylate.

[0007] Technical solution: The quality control method of melogabalin besylate described in the present invention comprises the following steps:

[0008] (1) preparing sample solution;

[0009] (2) Detection: High performance liquid chromatography was used, an octadecylsilane bonded silica gel column was used, a phosphate buffer solution-methanol mixed solution was used as mobile phase A, and an acetonitrile-phosphate buffer solution-methanol mixed solvent was used as mobile phase B. Gradient elution was performed according to the following procedure: the volume fraction of mobile phase A was reduced from 100% to 90% from 0 to 20 min, from 90% to 70% from 20 to 30 min, from 70% to 0% from 30 to 50 min, and maintained at 0% from 50 to 60 min;

[0010] (3) Analyze the content of each quality control impurity.

[0011] Preferably, the quality control impurities described in step (3) are as follows:

[0012]

[0013] Preferably, the gradient elution procedure described in step (2) is as follows:

[0014]

[0015] Further preferably, the gradient elution procedure is as follows:

[0016]

[0017]

[0018] Preferably, in the mobile phases A and B described in step (2), the phosphate buffer solution is a dipotassium hydrogen phosphate buffer solution with a concentration of 5 to 20 mmol / L and a pH of 5 to 7.

[0019] More preferably, the concentration of the dipotassium hydrogen phosphate buffer solution is 10 mmol / L and the pH is 6.0.

[0020] More preferably, the pH of the dipotassium hydrogen phosphate buffer solution is adjusted to 6.0 using phosphoric acid.

[0021] Preferably, in the mobile phase A described in step (2), the volume ratio of phosphate buffer solution to methanol is 1000:20; and in the mobile phase B, the volume ratio of acetonitrile, phosphate buffer solution and methanol is 640:200:160.

[0022] Preferably, the chromatographic column in step (2) has a length of 150 to 250 mm, a diameter of 4.6 mm, and a filler particle size of 3.5 to 5 μm.

[0023] More preferably, the chromatographic column has a length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.

[0024] More preferably, the chromatographic column is a Welch UltimateAQ-C18 chromatographic column or an equivalent chromatographic column, with a column length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.

[0025] Preferably, the detection wavelength of the detection in step (2) is 195-220 nm, the column temperature of the chromatographic column is 20-40° C., and the flow rate of the mobile phase is 1-1.5 ml / min.

[0026] More preferably, the detection wavelength is 215 nm and the column temperature of the chromatographic column is 30°C.

[0027] Preferably, the injection volume for the detection in step (2) is 25 μl.

[0028] Preferably, in the quality control method, the detection linear range of melogabalin besylate is 0.2994 to 3.9916 μg / ml.

[0029] More preferably, the detection linear equation of melogabalin besylate is y=0.1890x-0.0000.

[0030] Preferably, in the quality control method, the detection limit and quantification limit of each quality control component are as follows:

[0031]

[0032] Preferably, the sample solution described in step (1) is prepared using a phosphate buffer solution-methanol mixed solution with a volume ratio of 95:5.

[0033] More preferably, the concentration of the phosphate buffer solution is 10 mmol / ml and the pH is 6.0.

[0034] Further preferably, the sample solution comprises an impurity localization solution, a system suitability solution, and a test solution, and the specific preparation method is as follows:

[0035] Preparation of impurity localization solution: Take appropriate amount of reference substances of impurity 1, impurity 2, impurity 3, impurity 4 and impurity 5 respectively, weigh accurately, dissolve and dilute with diluent respectively, make solutions with the content of impurity 1 to impurity 5 of 3 μg per 1 ml, and use them as localization solutions of impurities 1 to impurity 5 respectively.

[0036] Preparation of system suitability solution: Take appropriate amounts of melogabalin besylate, impurity 1, impurity 2, impurity 3, impurity 4 and impurity 5, dissolve in diluent and quantitatively dilute to produce a solution containing 2 mg of melogabalin besylate and 3 μg of each of impurities 1 to 5 per 1 ml.

[0037] Preparation of test solution: Take an appropriate amount of melogabalin besylate, dissolve it in a diluent and quantitatively dilute it to make a solution containing 2 mg of melogabalin besylate per 1 ml.

[0038] Preferably, in step (3), the content of each quality control impurity is analyzed by the principal component self-control method with the addition of a correction factor.

[0039] Further preferably, the correction factors for each quality control impurity are as follows:

[0040]

[0041] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0042] The present invention optimizes chromatographic conditions for gradient elution, can effectively elute, separate and quantify a variety of specific impurities in melogabalin besylate, has strong method specificity, wide linear range, high sensitivity, good accuracy, and good solution stability and durability, and is beneficial to the quality control of melogabalin besylate raw materials and preparations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The HPLC detection chromatogram of the system suitability solution of Example 1;

[0044] Figure 2 The HPLC chromatogram of the test solution of Example 1;

[0045] Figure 3 It is the HPLC detection chromatogram of the system suitability solution of Comparative Example 1;

[0046] Figure 4 The HPLC detection chromatogram of the blank solution of Comparative Example 1;

[0047] Figure 5 The HPLC detection chromatogram of Comparative Example 2 is shown;

[0048] Figure 6 The HPLC detection chromatogram of Comparative Example 3 is shown. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below in conjunction with embodiments.

[0050] Example 1

[0051] 1. Experimental methods

[0052] Instrument: Thermo Ultimate 3000;

[0053] Chromatographic column: Welch Ultimate AQ-C18 4.6×250mm, 5μm;

[0054] Mobile phase: Phase A: 10 mmol / L dipotassium hydrogen phosphate solution (pH adjusted to 6.0 with phosphoric acid)-methanol (1000:20), Phase B: acetonitrile-10 mmol / L dipotassium hydrogen phosphate solution (pH adjusted to 6.0 with phosphoric acid)-methanol (640:200:160);

[0055] Detection wavelength: 215nm;

[0056] Column temperature: 30°C;

[0057] Injection volume: 25 μl.

[0058] Table 1 Gradient elution program and flow rate of Example 1

[0059]

[0060] Dilution solution (blank solution): 10 mmol / L potassium dihydrogen phosphate solution (pH 6.0)-methanol (95:5).

[0061] Preparation of impurity localization solution: Take appropriate amount of reference substances of impurity 1, impurity 2, impurity 3, impurity 4 and impurity 5 respectively, weigh accurately, dissolve and dilute with diluent respectively, make solutions with the content of impurity 1 to impurity 5 of 3 μg per 1 ml, and use them as localization solutions of impurities 1 to impurity 5 respectively.

[0062] Preparation of system suitability solution: Take appropriate amounts of melogabalin besylate, impurity 1, impurity 2, impurity 3, impurity 4 and impurity 5, dissolve in diluent and quantitatively dilute to make a solution containing approximately 2 mg melogabalin besylate and 3 μg of each impurity 1 to impurity 5 per 1 ml, shake well and set aside.

[0063] Preparation of test solution: Take an appropriate amount of melogabalin besylate, dissolve it in diluent and quantitatively dilute it to make a solution containing about 2 mg of melogabalin besylate per 1 ml, shake well and set aside.

[0064] Sample determination: Take 25 μl of blank solution, impurity location solution, system suitability solution and test solution respectively and inject them into the liquid chromatograph. The contents of the main components and impurities and their measured retention times are shown in Tables 2 and 3.

[0065] Table 2 Positioning detection results of Example 1

[0066] Components Impurity localization solution content (μg / ml) System suitability solution content (μg / ml) Retention time (min) principal component N / A 2000 35.405 Impurity 1 3 3 35.045 Impurity 2 3 3 34.325 Impurity 3 3 3 44.512 Impurity 4 3 3 16.925 Impurity 5 3 3 17.599

[0067] Table 3 Test results of the test solution of Example 1

[0068] Components Impurity content (%) Retention time (min) principal component N / A 35.404 Impurity 1 0.02 35.051 Impurity 2 0.04 34.304 Impurity 3 0.03 44.838 Impurity 4 0.05 16.904 Impurity 5 0.04 17.577

[0069] 2. Experimental results

[0070] Depend on Figure 1 It can be seen that RT = 35.405min is the peak of melogabalin benzenesulfonate, RT = 16.925min is the peak of impurity 4, RT = 17.599min is the peak of impurity 5, RT = 34.325min is the peak of impurity 2, RT = 35.045min is the peak of impurity 1, and RT = 44.512min is the peak of impurity 3. The main peak of melogabalin benzenesulfonate detected by this method can achieve baseline separation with the chromatographic peaks of adjacent impurities and known impurities, and the minimum separation degree is 1.71. Figure 2 It can be seen that the main peak of melogabalin besylate detected by this method can achieve baseline separation from the chromatographic peaks of adjacent impurities and known impurities, with a minimum separation degree of 2.22.

[0071] 3. Methodological validation

[0072] With reference to the "Guidelines for Validation of Analytical Methods", the quality control method of Example 1 was methodologically validated.

[0073] Table 4 Methodology validation results

[0074]

[0075]

[0076]

[0077] It can be seen from the above methodological verification results that the quality control method designed by the present invention has strong specificity, wide linear range, high sensitivity, high accuracy, good solution stability and durability, and can achieve effective detection of melogabalin besylate and its impurities, thus meeting the quality control requirements.

[0078] Comparative Example 1

[0079] 1. Experimental methods

[0080] Instrument: Thermo Ultimate 3000;

[0081] Column: MicroPulite T3 4.6×250mm, 5μm;

[0082] Mobile phase: Phase A: 10 mmol / L potassium dihydrogen phosphate solution (pH adjusted to 6.0 with phosphoric acid)-methanol (1000:20), Phase B: methanol-acetonitrile (30:70);

[0083] Detection wavelength: 215nm;

[0084] Flow rate: 1.0ml / min;

[0085] Column temperature: 30°C;

[0086] Injection volume: 5μl.

[0087] Table 5 Gradient elution program of Comparative Example 1

[0088]

[0089] Dilution solution (blank solution): acetonitrile-water (95:5).

[0090] Preparation of impurity localization solution: Take appropriate amount of reference substances of impurity 1, impurity 2, impurity 3, impurity 4 and impurity 5 respectively, weigh accurately, add diluent to dissolve and dilute respectively, to make solutions with the content of impurity 1 to impurity 5 of about 7.5 μg per 1 ml, which are used as localization solutions of impurities 1 to impurity 5 respectively.

[0091] Preparation of system suitability solution: Take appropriate amount of melogabalin besylate, impurity 1, impurity 2, impurity 3, impurity 4 and impurity 5 reference substances, add diluent to dissolve and quantitatively dilute to make a solution containing approximately 5 mg melogabalin besylate, approximately 7.5 μg impurity 1, approximately 7.5 μg impurity 2, approximately 7.5 μg impurity 3, approximately 7.5 μg impurity 4 and approximately 7.5 μg impurity 5 per 1 ml, shake well and set aside.

[0092] Sample determination: 5 μl of blank solution, impurity location solution and system suitability solution were measured and injected into the liquid chromatograph. The contents of the main components and impurities and their measured retention times are shown in Table 6.

[0093] Table 6 Positioning detection results of comparative example 1

[0094] Components Impurity localization solution content (μg / ml) System suitability solution content (μg / ml) Retention time (min) principal component N / A 5000 32.430 Impurity 1 7.5 7.5 30.907 Impurity 2 7.5 7.5 31.920 Impurity 3 7.5 7.5 44.813 Impurity 4 7.5 7.5 15.467 Impurity 5 7.5 7.5 16.047

[0095] 2. Experimental results

[0096] Depend on Figure 3 It can be seen that RT = 32.430min is the peak of melogabalin benzenesulfonate, RT = 15.467min is the peak of impurity 4, RT = 16.047min is the peak of impurity 5, RT = 30.907min is the peak of impurity 1, RT = 31.920min is the peak of impurity 2, RT = 44.813min is the peak of impurity 3, and the others are unknown impurity peaks. Figure 4 It can be seen that the baselines of impurities 4 and 5 detected by this method are raised at the peaks, forming bulges, which interfere with the detection and therefore cannot meet the quality control requirements.

[0097] Comparative Example 2

[0098] 1. Experimental methods

[0099] Instrument: Thermo Ultimate 3000;

[0100] Column: Thermo Hypersil Gold, 4.6 mm × 250 mm, 5 μm;

[0101] Mobile phase: Phase A: 10 mmol / L potassium dihydrogen phosphate solution (pH adjusted to 7.2 with phosphoric acid)-acetonitrile (95:5), Phase B: methanol-acetonitrile (50:50);

[0102] Detection wavelength: 215nm;

[0103] Flow rate: 1.0ml / min;

[0104] Column temperature: 30°C;

[0105] Injection volume: 5μl.

[0106] Table 7 Gradient elution program of Comparative Example 2

[0107]

[0108] Dilution solution (blank solution): acetonitrile-water (95:5).

[0109] Preparation of impurity localization solution: Take appropriate amount of reference substances of impurity 1, impurity 2, impurity 3, impurity 4 and impurity 5 respectively, weigh accurately, add diluent to dissolve and dilute respectively, to make solutions with the content of impurity 1 to impurity 5 of about 7.5 μg per 1 ml, which are used as localization solutions of impurities 1 to impurity 5 respectively.

[0110] Preparation of test solution: Take an appropriate amount of melogabalin besylate, dissolve it in diluent and quantitatively dilute it to make a solution containing about 5 mg of melogabalin besylate per 1 ml, shake well and set aside.

[0111] Sample determination: 5 μl of blank solution, impurity location solution and test solution were measured and injected into the liquid chromatograph. The contents of the main components and impurities and their measured retention times are shown in Table 8.

[0112] Table 8 Positioning detection results of comparative example 2

[0113] Components Impurity localization solution content (μg / ml) System suitability solution content (μg / ml) Retention time (min) principal component N / A 5000 19.003 Impurity 1 7.5 7.5 17.420 Impurity 2 7.5 7.5 18.417 Impurity 3 7.5 7.5 31.677 Impurity 4 7.5 7.5 6.517 Impurity 5 7.5 7.5 6.883

[0114] 2. Experimental results

[0115] Depend on Figure 5 It can be seen that RT = 19.003min is the peak of melogabalin benzenesulfonate, RT = 6.517min is the peak of impurity 4, RT = 6.883min is the peak of impurity 5, RT = 17.420min is the peak of impurity 1, RT = 18.417min is the peak of impurity 2, RT = 31.677min is the peak of impurity 3, and the others are all unknown impurity peaks. This method does not effectively separate impurity 4, impurity 5 and benzenesulfonic acid peaks (separation degree <1.5), so it cannot meet the quality control requirements.

[0116] Comparative Example 3

[0117] 1. Experimental methods

[0118] Instrument: Thermo Ultimate 3000;

[0119] Chromatographic column: Welch Ultimate AQ-C18 4.6×250mm, 5μm;

[0120] Mobile phase: Phase A: 10 mmol / L dipotassium hydrogen phosphate solution (pH adjusted to 6.0 with phosphoric acid)-methanol (1000:20), Phase B: acetonitrile-10 mmol / L dipotassium hydrogen phosphate solution (pH adjusted to 6.0 with phosphoric acid)-methanol (640:200:160);

[0121] Detection wavelength: 215nm;

[0122] Column temperature: 30°C;

[0123] Injection volume: 25 μl.

[0124] Table 9 Gradient elution program and flow rate of comparative example 3

[0125]

[0126] Dilution solution (blank solution): acetonitrile-water (50:50).

[0127] Preparation of test solution: Take an appropriate amount of melogabalin besylate, dissolve it in a diluent and quantitatively dilute it to make a solution containing about 2 mg of melogabalin besylate per 1 ml, and set aside.

[0128] 2. Experimental results

[0129] Depend on Figure 6 It can be seen that RT = 34.387min and RT = 34.623min are both peaks of melogabalin benzenesulfonate. The melogabalin benzenesulfonate peak of this method is bifurcated, so it cannot meet the quality control requirements.

Claims

1. A quality control method for melogabalin besylate, characterized in that, The following steps are involved: (1) preparing sample solution; (2) Detection: High performance liquid chromatography was used, an octadecylsilane bonded silica gel column was used, a phosphate buffer solution-methanol mixed solution was used as mobile phase A, and an acetonitrile-phosphate buffer solution-methanol mixed solvent was used as mobile phase B. Gradient elution was performed according to the following procedure: based on the volume fraction of mobile phase A, 0-20 min: decreased from 100% to 90%, 20-30 min: decreased from 90% to 70%, 30-50 min: decreased from 70% to 0%, 50-60 min: maintained at 0%; (3) Analyze the content of each quality control impurity.

2. The quality control method according to claim 1, characterized in that: The quality control impurities described in step (3) are as follows:

3. The quality control method according to claim 1, characterized in that: The gradient elution procedure described in step (2) is as follows:

4. The quality control method according to claim 1, characterized in that: In the mobile phases A and B described in step (2), the phosphate buffer solution is a dipotassium hydrogen phosphate buffer solution with a concentration of 5 to 20 mmol / L and a pH of 5 to 7.

5. The quality control method according to claim 4, characterized in that: The concentration of the dipotassium hydrogen phosphate buffer solution is 10 mmol / L, and the pH is 6.

0.

6. The quality control method according to claim 1, characterized in that: In the mobile phase A described in step (2), the volume ratio of phosphate buffer solution to methanol is 1000:20; in the mobile phase B, the volume ratio of acetonitrile, phosphate buffer solution and methanol is 640:200:

160.

7. The quality control method according to claim 1, characterized in that: The column length of the chromatographic column in step (2) is 150-250 mm, the diameter is 4.6 mm, and the filler particle size is 3.5-5 μm.

8. The quality control method according to claim 7, characterized in that: The chromatographic column has a length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.

9. The quality control method according to claim 1, characterized in that: The detection wavelength of the detection in step (2) is 195-220 nm, the column temperature of the chromatographic column is 20-40° C., and the flow rate of the mobile phase is 1-1.5 ml / min.

10. The quality control method according to claim 1, characterized in that: The sample solution described in step (1) is prepared by using a phosphate buffer solution-methanol mixed solution with a volume ratio of 95:5.

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