Method for separating and determining remegapam intermediate related substances by HPLC (High Performance Liquid Chromatography)

Abstract

The invention provides a method for separating and determining related substances of a remegapam intermediate by using HPLC (High Performance Liquid Chromatography), which comprises the following steps: carrying out gradient elution by using octadecylsilane chemically bonded silica as a chromatographic column filler, 0.01 mol / L potassium hexafluorophosphate solution as a mobile phase A and a solution obtained by uniformly mixing methanol and acetonitrile according to a volume ratio of (55-65): (35-45) as a mobile phase B at the flow velocity of 0.8-1.2 ml / min and the column temperature of 25-40 DEG C, the method comprises the following steps: detecting related substances of the remegapam intermediate by adopting an ultraviolet detector, wherein the detection wavelength of the ultraviolet detector is 260-270nm; the related substances of the remegapam intermediate are one or more of an impurity I, an impurity II and an impurity III. According to the present invention, the impurity I, the impurity II and the impurity III in the remegapam intermediate can be rapidly and efficiently separated under the same chromatographic condition so as to effectively control the quality of the bulk drug, and the detection method has advantages of rapidness, simpleness, convenience, high sensitivity, strong specificity, high precision, accuracy, reliability and convenient operation.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for separating and determining related substances of a remegipam intermediate by using HPLC. Background Art

[0002] Rimegepant, also known as BMS-927711, NURTECTMODT, is an effective, selective, competitive, orally active calcitonin gene-related peptide (CGRP) antagonist that can be used for both acute and preventive treatment of migraine. On February 27, 2020, it was approved for marketing by the U.S. Food and Drug Administration (FDA). It is used for the acute treatment of migraine in adults. The molecular formula is C 28 H 28 F2N6O3, molecular weight is 534.56, structural formula is shown in formula 1:

[0003]

[0004] Remigipam A-4 is an intermediate of Remigipam, with the molecular formula of C 10 H 11 NO2, molecular weight: 177.20, chemical structure is shown in Formula 2:

[0005]

[0006] During the preparation process of the Remigipam intermediate, multiple impurities are generated due to various factors such as starting materials, synthesis process, and degradation. Among them, impurities I, II, and III are more easily produced in the synthesis process and are the main impurities investigated in the related substance items. The limit requirement for them is not more than 0.50wt%.

[0007] Impurity I is the starting material, molecular formula: C 10 H9NO2, molecular weight: 175.19, chemical structure is shown in formula 3:

[0008]

[0009] Impurity II is a by-product, molecular formula: C 10 H 13 NO2, molecular weight: 179.22, chemical structure is shown in formula 4:

[0010]

[0011] Impurity III is a by-product, molecular formula: C 10 H 13 NO2, molecular weight: 179.22, chemical structure is shown in Formula 5:

[0012]

[0013] Currently, no method has been found that can quickly and efficiently separate impurities I, II and III in the intermediate of Remigipam under the same chromatographic conditions.

[0014] Based on this, the present invention is proposed. Summary of the invention

[0015] In order to overcome the above technical problems existing in the prior art, the inventors, after conducting a lot of in-depth research, provide a method for separating and determining related substances of remegipam intermediates by HPLC, which has the advantages of being rapid, simple, highly sensitive, accurate and reliable.

[0016] The technical solution adopted by the present invention is:

[0017] A method for separating and determining related substances of a remegipam intermediate by HPLC comprises the following steps:

[0018] Chromatographic conditions:

[0019] Chromatographic column: Octadecylsilane bonded silica gel is the chromatographic column filler;

[0020] A 0.01 mol / L potassium hexafluorophosphate solution is used as mobile phase A, and methanol and acetonitrile are mixed in a volume ratio of (55-65):(35-45) to obtain a solution as mobile phase B for gradient elution. The flow rate is 0.8-1.2 ml / min, the column temperature is 25-40° C., and the related substances of the remigipam intermediate are detected by an ultraviolet detector; the detection wavelength of the ultraviolet detector is 260-270 nm.

[0021] The related substances of the remegipam intermediate are one or more of impurities I, II and III.

[0022] Furthermore, the column length of the chromatographic column is 150 mm to 250 mm; preferably, the column length of the chromatographic column is 250 mm.

[0023] Furthermore, the particle size of the chromatographic column filler is 1.8 to 5 μm; preferably, the particle size of the chromatographic column filler is 5 μm.

[0024] Furthermore, the procedure of the gradient elution is:

[0025] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 85～75 15～25 15 55～45 45～55 25 55～45 45～55 25.01 85～75 15～25 35 85～75 15～25

[0026] Furthermore, the procedure of the gradient elution is:

[0027] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 80 20 15 50 50 25 50 50 25.01 80 20 35 80 20

[0028] This results in the best separation and the best peak shape.

[0029] Furthermore, the flow rate is 1.0 ml / min and the column temperature is 35° C. Thus, the separation degree between impurities can be improved.

[0030] Furthermore, the detection wavelength of the ultraviolet detector is 267 nm, thereby improving the detection sensitivity of impurities.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The invention optimizes the detection result by comprehensively considering the comprehensive influence of the analytical column, the mobile phase, the gradient elution program, the flow rate and the column temperature on the separation and detection, and can quickly and efficiently separate the impurities I, II and III in the remigipam intermediate under the same chromatographic conditions, thereby effectively controlling the quality of the raw material drug, and the detection method has the advantages of being rapid and simple, highly sensitive, highly specific, highly precise, accurate and reliable, and easy to operate, and is suitable for separating and determining the related substances of the remigipam intermediate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The chromatogram is a blank solution detected under the conditions of Example 1 in the present invention;

[0034] Figure 2 The chromatogram of the system suitability solution tested under the conditions of Example 1 in the present invention;

[0035] Figure 3 The chromatogram of the test solution detected according to the conditions of Example 1 in the present invention;

[0036] Figure 4 The chromatogram is a quantitative limit solution detected according to the conditions of Example 1 in the present invention;

[0037] Figure 5 The chromatogram is a detection limit solution detected under the conditions of Example 1 in the present invention;

[0038] Figure 6 The chromatogram is a blank solution detected under the conditions of Example 2 in the present invention;

[0039] Figure 7 The chromatogram of the system suitability solution tested under the conditions of Example 2 in the present invention;

[0040] Figure 8 The chromatogram of the test solution detected according to the conditions of Example 2 in the present invention;

[0041] Fig. 9 The chromatogram is a quantitative limit solution detected under the conditions of Example 2 in the present invention;

[0042] Fig.10This is a chromatogram of the detection limit solution detected under the conditions of Example 2 in the present invention.

[0043] Fig.11 The chromatogram is a blank solution detected under the conditions of Example 3 in the present invention;

[0044] Fig.12 The chromatogram of the system suitability solution tested under the conditions of Example 3 in the present invention;

[0045] Fig.13 The chromatogram of the test solution detected according to the conditions of Example 3 in the present invention;

[0046] Fig.14 The chromatogram is a quantitative limit solution detected under the conditions of Example 3 in the present invention;

[0047] Fig.15 This is a chromatogram of the detection limit solution detected under the conditions of Example 3 in the present invention. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with specific embodiments and accompanying drawings. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some variations and improvements can also be made without departing from the inventive concept. These all belong to the protection scope of the present invention. Reagents and instruments used that do not indicate the manufacturer, all can be obtained through commercial conventional products.

[0049] The remigipam intermediates and impurity reference substances used in the examples of the present invention were all prepared by the inventors themselves.

[0050] Example 1

[0051] The chromatographic conditions were as follows:

[0052] Chromatographic column: Agilent TC-C18 (4.6 mm × 250 mm, 5 μm);

[0053] Mobile phase A: 0.01 M potassium hexafluorophosphate solution;

[0054] Mobile phase B: methanol-acetonitrile (55:45) (v / v);

[0055] Column temperature: 35°C;

[0056] Flow rate: 1.0ml / min;

[0057] Detection wavelength of UV detector: 267nm;

[0058] Injection volume: 10 μl;

[0059] The gradient elution program was:

[0060] Table 1 Gradient elution program

[0061] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 80 20 15 50 50 25 50 50 25.01 80 20 35 80 20

[0062] Solution preparation:

[0063] Impurity reference substance stock solution: Take about 20 mg of each of the impurity I reference substance, the impurity II reference substance and the impurity III reference substance, accurately weigh, place in the same 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: 50% acetonitrile aqueous solution.

[0064] System suitability solution: Take about 50 mg of Remigipam A4 working reference substance, accurately weigh it, and place it in a 50 ml volumetric flask. Accurately add 0.5 ml of the above impurity reference substance stock solution into the above 50 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0065] Test solution: Take about 25 mg of Remigipam intermediate, weigh accurately, place in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0066] Remigipam A4 reference substance stock solution: Take about 25 mg of Remigipam A4 working reference substance, accurately weigh it, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0067] Quantitation limit solution: Accurately measure 1 ml of the above impurity reference stock solution and Remigipan A4 reference stock solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, then accurately measure 4 ml to 100 ml, add diluent to dilute to the scale, shake well, and obtain.

[0068] Detection limit solution: Accurately measure 1 ml of the above impurity reference stock solution and Remigipan A4 reference stock solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, then accurately measure 2 ml to 100 ml, add diluent to dilute to the scale, shake well, and obtain.

[0069] Determination: Inject the blank solution (i.e. diluent), system suitability solution, quantitative limit solution, detection limit solution and test solution into the high performance liquid chromatograph for detection respectively. Use octadecylsilane bonded silica gel as the chromatographic column filler (particle size is 5 μm, column inner diameter is 4.6 mm, chromatographic column length is 250 mm). Detect according to the gradient elution program in Table 1 and record the chromatogram.

[0070] The chromatograms of blank solution, system suitability solution, quantitation limit solution, detection limit and test solution are shown in Figure 1. Figure 1 , 2 , 3, 4 and 5, it can be seen that Figure 1This indicates that the blank solution does not interfere with the determination of impurities; Figure 2 It shows that the separation between each impurity and Remigipam A4 is good, and the specific data are shown in Table 2; Figure 3 The results show that the quantitative limits of Remigipam A4 and impurity I, impurity II and impurity III are 0.04wt%, 0.04wt% and 0.04wt%, respectively, which are lower than the limit of 0.10wt% of each impurity. The detection sensitivity of the method in this embodiment is relatively high. Figure 5 Impurity II was detected in the homemade Remigipam A4 sample, while impurity I and impurity III were not detected. Other individual impurities detected were all below the prescribed limits.

[0071] Table 2 System suitability data table in Example 1

[0072] Example 2

[0073] The chromatographic conditions were as follows:

[0074] Chromatographic column: Agilent TC-C18 (4.6 mm × 250 mm, 5 μm);

[0075] Mobile phase A: 0.01 M potassium hexafluorophosphate solution;

[0076] Mobile phase B: methanol-acetonitrile (60:40) (v / v);

[0077] Column temperature: 35°C;

[0078] Flow rate: 1.0ml / min;

[0079] Detection wavelength of UV detector: 267nm;

[0080] Injection volume: 10 μl;

[0081] The gradient elution program was:

[0082] Table 3 Gradient elution program

[0083]

[0084]

[0085] Solution preparation:

[0086] Impurity reference substance stock solution: Take about 20 mg of each of the impurity I reference substance, the impurity II reference substance and the impurity III reference substance, accurately weigh, place in the same 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: 50% acetonitrile aqueous solution.

[0087] System suitability solution: Take about 50 mg of Remigipam A4 working reference substance, accurately weigh it, and place it in a 50 ml volumetric flask. Accurately add 0.5 ml of the above impurity reference substance stock solution into the above 50 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0088] Test solution: Take about 25 mg of Remigipam intermediate, weigh accurately, place in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0089] Remigipam A4 reference substance stock solution: Take about 25 mg of Remigipam A4 working reference substance, accurately weigh it, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0090] Quantitation limit solution: Accurately measure 1 ml of the above impurity reference stock solution and Remigipan A4 reference stock solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, then accurately measure 4 ml to 100 ml, add diluent to dilute to the scale, shake well, and obtain.

[0091] Detection limit solution: Accurately measure 1 ml of the above impurity reference stock solution and Remigipan A4 reference stock solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, then accurately measure 2 ml to 100 ml, add diluent to dilute to the scale, shake well, and obtain.

[0092] Determination: Inject the blank solution (i.e. diluent), system suitability solution, quantitative limit solution, detection limit solution and test solution into the high performance liquid chromatograph for detection respectively. Use octadecylsilane bonded silica gel as the chromatographic column filler (particle size is 5 μm, column inner diameter is 4.6 mm, chromatographic column length is 250 mm). Detect according to the gradient elution program in Table 3 and record the chromatogram.

[0093] The chromatograms of blank solution, system suitability solution, quantitation limit solution, detection limit and test solution are shown in Figure 1. Figure 6 , 7 , 8, 9 and 10, it can be seen that Figure 7 It shows that the separation between impurities and Remigipam A4 is good, and the specific data are shown in Table 4; Fig.10 Impurity II was detected in the homemade Remigipam A4 sample, while impurity I and impurity III were not detected. The other individual impurities detected were all below the specified limits. Example 2, based on the method of Example 1, mainly investigated the separation test between impurities and Remigipam A4 (i.e., system suitability test) and compared the impurity spectrum of the test solution with that of Example 1, and the results were basically consistent.

[0094] Table 4 System suitability data table in Example 2 Example 3

[0095] The chromatographic conditions were as follows:

[0096] Chromatographic column: Agilent TC-C18 (4.6 mm × 250 mm, 5 μm);

[0097] Mobile phase A: 0.01 M potassium hexafluorophosphate solution;

[0098] Mobile phase B: methanol-acetonitrile (65:35) (v / v);

[0099] Column temperature: 35°C;

[0100] Flow rate: 1.0ml / min;

[0101] Detection wavelength of UV detector: 267nm;

[0102] Injection volume: 10 μl;

[0103] The gradient elution program was:

[0104] Table 5 Gradient elution program

[0105] Time, minutes Mobile phase A, volume % Mobile phase B, volume % 0 80 20 15 50 50 25 50 50 25.01 80 20 35 80 20

[0106] Solution preparation:

[0107] Impurity reference substance stock solution: Take about 20 mg of each of the impurity I reference substance, the impurity II reference substance and the impurity III reference substance, accurately weigh, place in the same 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain; the diluent is: 50% acetonitrile aqueous solution.

[0108] System suitability solution: Take about 50 mg of Remigipam A4 working reference substance, accurately weigh it, and place it in a 50 ml volumetric flask. Accurately add 0.5 ml of the above impurity reference substance stock solution into the above 50 ml volumetric flask, then add diluent to dilute to the scale, shake well, and obtain.

[0109] Test solution: Take about 25 mg of Remigipam intermediate, weigh accurately, place in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0110] Remigipam A4 reference substance stock solution: Take about 25 mg of Remigipam A4 working reference substance, accurately weigh it, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0111] Quantitation limit solution: Accurately measure 1 ml of the above impurity reference stock solution and Remigipan A4 reference stock solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, then accurately measure 4 ml to 100 ml, add diluent to dilute to the scale, shake well, and obtain.

[0112] Detection limit solution: Accurately measure 1 ml of the above impurity reference stock solution and Remigipan A4 reference stock solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, then accurately measure 2 ml to 100 ml, add diluent to dilute to the scale, shake well, and obtain.

[0113] Determination: Inject the blank solution (i.e. diluent), system suitability solution, quantitative limit solution, detection limit solution and test solution into the high performance liquid chromatograph for detection respectively. Use octadecylsilane bonded silica gel as the chromatographic column filler (particle size is 5 μm, column inner diameter is 4.6 mm, chromatographic column length is 250 mm). Detect according to the gradient elution program in Table 5 and record the chromatogram.

[0114] The chromatograms of blank solution, system suitability solution, quantitation limit solution, detection limit and test solution are shown in Figure 1. Fig.11 , 12 , 13, 14 and 15, it can be seen that Fig.12 It shows that the separation between impurities and Remigipam A4 is good, and the specific data are shown in Table 6; Fig.15 Impurity II was detected in the homemade Remigipam A4 sample, while impurity I and impurity III were not detected. The other individual impurities detected were all below the specified limits. Example 3, based on the method of Example 1, mainly investigated the separation test between impurities and Remigipam A4 (i.e., system suitability test) and compared the impurity spectrum of the test solution with that of Example 1, and the results were basically consistent.

[0115] Table 6 System suitability data table in Example 3

[0116]

[0117] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for separating and determining related substances of remegipam intermediates by HPLC, characterized in that: The steps include: Chromatographic conditions: Chromatographic column: Octadecylsilane bonded silica gel is the chromatographic column filler; Using 0.01 mol / L potassium hexafluorophosphate solution as mobile phase A, methanol and acetonitrile in a volume ratio of (55-65): (35-45) as the mobile phase B for gradient elution, the flow rate is 0.8-1.2 ml / min, the column temperature is 25-40° C., and the related substances of the remigipam intermediate are detected by an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 260-270 nm; The related substance of the remegipam intermediate is one or more of impurities I, II and III; The structural formula of impurity I is shown in Formula 3: The structural formula of impurity II is shown in Formula 4: The structural formula of impurity III is shown in Formula 5:

2. The method for separating and determining related substances of remegipam intermediates by HPLC according to claim 1, characterized in that: The column length of the chromatographic column is 150 mm to 250 mm.

3. The method for separating and determining related substances of Remigipam intermediates by HPLC according to claim 1, characterized in that: The column length of the chromatographic column is 250 mm.

4. The method for separating and determining related substances of Remigipam intermediates by HPLC according to claim 1, characterized in that: The particle size of the chromatographic column filler is 1.8 to 5 μm.

5. The method for separating and determining related substances of remegipam intermediates by HPLC according to claim 1, characterized in that: The particle size of the chromatographic column filler is 5 μm.

6. The method for separating and determining related substances of Remigipam intermediates by HPLC according to claim 1, characterized in that: The procedure of the gradient elution is: 。 7. The method for separating and determining related substances of Remigipam intermediates by HPLC according to claim 1, characterized in that: The procedure of the gradient elution is: 。 8. The method for separating and determining related substances of Remigipam intermediates by HPLC according to claim 1, characterized in that: The flow rate was 1.0 ml / min and the column temperature was 35°C.

9. A method for separating and determining related substances of remegipam intermediates by HPLC according to any one of claims 1 to 8, characterized in that: The detection wavelength of the ultraviolet detector is 267 nm.

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