Determination method of nikethamide related substances

Abstract

The invention relates to a method for determining nikethamide related substances, which adopts reversed-phase high-performance liquid chromatography for gradient elution to separate nikethamide from related impurities. The determination method provided by the invention is high in sensitivity, strong in specificity, good in accuracy and simple, convenient and rapid to operate, perfects the quality standard of determination of the related substances in nikethamide, can perform strict quality control on the related substances in nikethamide, ensures the safety and reliability of nikethamide, and has practical significance.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for determining nikethamide-related substances. Background Art

[0002] Nikethamide is used for central respiratory and circulatory failure, poisoning by anesthetics and other central nervous system depressants.

[0003] It can selectively excite the respiratory center in the medulla oblongata, and can also reflexively excite the respiratory center through the carotid body and aortic body chemoreceptors, making breathing deeper and faster. Its excitatory effect is more obvious when the respiratory center is suppressed. It is mainly used clinically for respiratory and circulatory failure caused by diseases or central nervous system depressant poisoning. It has a significant effect on respiratory failure caused by cor pulmonale and respiratory depression caused by morphine overdose, and its rescue effect is second only to that of inhaled anesthetic poisoning.

[0004] Impurity level is one of the important factors for drug safety and efficacy, so how to accurately determine the content of impurities in nikethamide has become an urgent problem to be solved.

[0005] At present, nikethamide and nikethamide injection are included in Part II of the 2020 edition of the Chinese Pharmacopoeia, and are not included in other foreign pharmacopoeias. Currently, the Chinese Pharmacopoeia does not specify the limits for individual impurities, only the limits for total impurities.

[0006] In order to effectively analyze the quality of drugs and ensure the safety of drug use, it is necessary to develop a method for impurity determination that is highly sensitive, specific, durable, convenient, and effective. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a method for determining related substances of nikethamide, comprehensively analyze the impurity spectrum in nikethamide, and effectively separate nikethamide from various impurities. The method has high sensitivity, good specificity, high accuracy, and good durability, and can strictly control the quality of related substances in nikethamide, thereby ensuring the safety and reliability of nikethamide, and has practical significance.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] A method for determining nikethamide-related substances, using reverse phase high performance liquid chromatography, using octadecyl bonded silica gel as a filler in a chromatographic column, using 20 mmol potassium dihydrogen phosphate aqueous solution as a mobile phase A, using methanol as a mobile phase B, performing gradient elution, and separating nikethamide and related impurities;

[0010] The pH of mobile phase A was adjusted to 2.8 with phosphoric acid;

[0011] The relevant impurities are one or more of impurities SY012-01, SY012-01A, SY012-01B, SY012-01C, SY012-01D, SY012-A, SY012-B, SY012-C, SY012-D, and SY012-E;

[0012] The specific structural formula of the relevant impurities is as follows:

[0013]

[0014] Preferably, the gradient elution program is as follows:

[0015]

[0016] Preferably, the chromatographic column is one of COSMOSIL C18.

[0017] Preferably, the inner diameter of the chromatographic column is 4.6 mm, the length is 250 mm, and the filler particle size is 5 μm.

[0018] Preferably, the column temperature of the chromatographic column is 20-30°C, preferably 25°C.

[0019] Preferably, the injection volume for gradient elution is 10 μL to 50 μL, preferably 25 μL.

[0020] Preferably, the flow rate of gradient elution is 0.8 to 1.2 mL / min, preferably 1.0 mL / min.

[0021] Preferably, ultraviolet light detection is used with a wavelength of 262 to 265 nm, preferably 263 nm.

[0022] Preferably, the method comprises the following steps:

[0023] (1) Prepare the test solution: take the product and dilute it with water to make a solution containing about 4 μg of nikethamide per 1 mL; prepare the control solution: accurately measure an appropriate amount of the test solution and quantitatively dilute it with water to make a solution containing about 8 μg of nikethamide per 1 mL;

[0024] (2) Separation: using reverse phase high performance liquid chromatography, the chromatographic column is filled with octadecyl bonded silica gel, 20 mmol potassium dihydrogen phosphate aqueous solution is used as mobile phase A, methanol is used as mobile phase B, the mobile phase A is adjusted to pH 2.8 with phosphoric acid, and the test solution and the control solution obtained in step (1) are subjected to gradient elution;

[0025] (3) Detection: Use ultraviolet light with a wavelength of 262 to 265 nm, preferably 263 nm.

[0026] Result calculation: calculated by principal component self-control method with correction factor added.

[0027] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0028] 1. The determination method of the present invention comprehensively analyzes the impurity spectrum in nikethamide according to the synthesis process of nikethamide raw materials, and can effectively separate nikethamide from various impurities;

[0029] 2. The determination method of the present invention has high sensitivity, strong specificity, good accuracy, and is simple and fast to operate. It improves the quality standard for the determination of related substances of nikethamide, can perform strict quality control on the related substances in nikethamide, ensures the safety and reliability of nikethamide, and has practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The UV absorption spectra of some compounds in Example 1;

[0031] Figure 2 It is the detection spectrum of different mobile phase screening in Example 2; wherein, the chromatographic peak numbers marked in each chromatogram correspond to the impurity names as follows: impurity 1: SY012-01B; impurity 2: SY012-01A; impurity 3: SY012-01; impurity 4: SY012-01C; impurity 5: SY012-B; impurity 6: SY012-A; impurity 7: SY012-01D; impurity 8: SY012-C; impurity 9: SY012-E; impurity 10: main component peak; impurity 11: SY012-D;

[0032] Figure 3 It is the detection spectrum of different solvent screening in Example 3; wherein, the chromatographic peak numbers marked in each chromatogram correspond to the impurity names as follows: impurity 1: SY012-01B; impurity 2: SY012-01A; impurity 3: SY012-01; impurity 4: SY012-01C; impurity 5: SY012-B; impurity 6: SY012-A; impurity 7: SY012-01D; impurity 8: SY012-C; impurity 9: SY012-E; impurity 10: main component peak; impurity 11: SY012-D;

[0033] Figure 4It is the detection spectrum of different gradient screening in Example 4; wherein, the chromatographic peak numbers marked in each chromatogram correspond to the impurity names as follows: impurity 1: SY012-01B; impurity 2: SY012-01A; impurity 3: SY012-01; impurity 4: SY012-01C; impurity 5: SY012-B; impurity 6: SY012-A; impurity 7: SY012-01D; impurity 8: SY012-C; impurity 9: SY012-E; impurity 10: main component peak; impurity 11: SY012-D;

[0034] Figure 5 is the ultraviolet absorption spectrum of each compound in Example 5;

[0035] Figure 6 It is the detection spectrum of different chromatographic columns screened in Example 6; wherein, the chromatographic peak numbers marked in each chromatogram correspond to the impurity names as follows: impurity 1: SY012-01B; impurity 2: SY012-01A; impurity 3: SY012-01; impurity 4: SY012-01C; impurity 5: SY012-B; impurity 6: SY012-A; impurity 7: SY012-01D; impurity 8: SY012-C; impurity 9: SY012-E; impurity 10: main component peak; impurity 11: SY012-D;

[0036] Figure 7 It is the detection spectrum of different column temperature screening in Example 7; wherein, the chromatographic peak numbers marked in each chromatogram correspond to the impurity names as follows: impurity 1: SY012-01B; impurity 2: SY012-01A; impurity 3: SY012-01; impurity 4: SY012-01C; impurity 5: SY012-B; impurity 6: SY012-A; impurity 7: SY012-01D; impurity 8: SY012-C; impurity 9: SY012-E; impurity 10: main component peak; impurity 11: SY012-D;

[0037] Figure 8 It is the detection spectrum of different mobile phase pH screening in Example 8; wherein, the chromatographic peak numbers marked in each chromatogram correspond to the impurity names as follows: impurity 1: SY012-01B; impurity 2: SY012-01A; impurity 3: SY012-01; impurity 4: SY012-01C; impurity 5: SY012-B; impurity 6: SY012-A; impurity 7: SY012-01D; impurity 8: SY012-C; impurity 9: SY012-E; impurity 10: main component peak; impurity 11: SY012-D;

[0038] Fig. 9 This is a comparison chart of the specific property test in Example 9;

[0039] Fig.10 This is the detection spectrum of the stability of the system suitability solution in Example 15. DETAILED DESCRIPTION

[0040] In order to make the technical solutions and beneficial effects of the present invention more obvious and easy to understand, the following is a detailed description by combining the drawings and listing specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Any product that is the same or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts falls within the protection scope of the present invention. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meaning as the technical and scientific terms in the technical field to which this application belongs.

[0041] The experimental methods in the following examples without specifying specific conditions are usually based on conventional experimental conditions.

[0042] The nikethamide, equipment, and reference substance used in the specific implementation manner of the present invention are all known products, and nikethamide, equipment, and reference substance are obtained by purchasing commercially available products.

[0043] Table 1 Equipment information

[0044]

[0045]

[0046] Table 2 Material information

[0047] name source batch number Ultrapure water Milli-Q Direct8 Homemade / Potassium dihydrogen phosphate Shanghai Lingfeng Chemical Reagent Co., Ltd. 20211201 Methanol TEDIA 21045193 Phosphoric acid Sinopharm Chemical Reagent Co., Ltd. 20200731 Niclosamide raw materials Lu'an Huayuan Pharmaceutical Co., Ltd. 21041901

[0048] Table 3 Impurity reference substance information

[0049]

[0050] Example 1

[0051] Solution preparation:

[0052] Solvent: Water

[0053] Each impurity localization solution: accurately weigh appropriate amount of impurity SY012-01A, SY012-01B, SY012-01C, SY012-C, and SY012-D reference substances, dissolve and dilute with solvent to make a 0.1 mg / mL solution, shake well, and use as each impurity localization solution.

[0054] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0055] Chromatographic column: Model: Thermo Scientific Hypersil Gold; Specifications: inner diameter 4.6 mm, length 250 mm, filler particle size 5 μm;

[0056] Mobile phase: Mobile phase A was 20 mmol ammonium acetate aqueous solution (pH adjusted to 5.0 with glacial acetic acid), and mobile phase B was methanol;

[0057] The gradient elution program is as follows:

[0058]

[0059] Detection wavelength: 190~400nm;

[0060] Column temperature: 30°C;

[0061] Flow rate: 1.0 mL / min;

[0062] Injection volume: 10 μL;

[0063] The UV absorption spectra of each compound were plotted in the wavelength range of 190-400 nm. The test results are shown in the attached Figure 1 . Impurity SY012-01A has maximum absorption peaks at 265.52nm and 212.66nm, impurity SY012-01B has maximum absorption peaks at 265.22nm and 208.33nm, impurity SY012-01C has maximum absorption peaks at 263.49nm and 208.86nm, SY012-C has maximum absorption peaks at 262.55nm and 211.84nm, and SY012-D has maximum absorption peaks at 262.03nm and 209.83nm. Each impurity has maximum absorption at 262.02nm~265.51nm and at the end. Referring to the detection wavelength of 263nm under the related substance detection item of nikethamide injection in Part II of the 2020 edition of the Chinese Pharmacopoeia, the tentative detection wavelength is 263nm.

[0064] Example 2

[0065] Solution preparation:

[0066] Solvent: Water

[0067] Stock solutions of each impurity: Accurately weigh about 8 mg of each of the impurity SY012-01, SY012-01A, SY012-01B, SY012-01C, SY012-01D, SY012-A, SY012-B, SY012-C, SY012-D, and SY012-E reference substances, place them in a 10 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0068] Test sample stock solution: Accurately weigh about 4 mg of the test sample and place it in a 10 mL volumetric flask. Add solvent to dissolve and dilute to the scale. Shake well to obtain.

[0069] Mixed solution: Accurately measure 100 μL of the test solution and each impurity stock solution, place them in the same 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0070] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions in Table 4 below were respectively adopted:

[0071] Table 4 Screening of different mobile phases

[0072]

[0073]

[0074] The test results are attached. Figure 2 : In the chromatogram of the mixed solution of method 1, the peaks of impurities SY012-01A and SY012-01C are poor, but the other 8 impurities can be effectively separated; in the chromatogram of the mixed solution of method 2, the impurity SY012-E overlaps with the main component peak, and the chromatographic conditions are not suitable; in the chromatogram of the mixed solution of method 3, the impurity SY012-E overlaps with the main component peak, and the impurity SY012-01C overlaps with the impurity SY012-D, and the chromatographic conditions are not suitable. It is tentatively decided to use method 1, that is, mobile phase pH 3.0, to further explore different solvents.

[0075] However, under the conditions of method 3, the peak shape of impurity SY012-01C was normal, and it was inferred that the peak shape of SY012-01C was good under neutral or alkaline mobile phase conditions.

[0076] Example 3

[0077] Solution preparation:

[0078] Solvent 1: Water

[0079] Solvent 2: Mobile phase A

[0080] The impurity stock solutions and test sample stock solutions were prepared as in Example 2.

[0081] Impurity SY012-01A / 01C positioning solution - water: Accurately measure 100 μL of impurity SY012-01A and impurity SY012-01C stock solutions, place them in the same 10 mL volumetric flask, add solvent 1 to dilute to the scale, shake well, and obtain.

[0082] Impurity SY012-01A / 01C positioning solution - mobile phase: Accurately measure 100 μL of impurity SY012-01A and impurity SY012-01C stock solutions, place them in the same 10 mL volumetric flask, add solvent 2 to dilute to the scale, shake well, and obtain.

[0083] Test sample + other 8 impurity mixed solution: Accurately measure 100 μL each of the test sample stock solution and the other 8 impurity stock solutions, place them in the same 10 mL volumetric flask, add solvent 1 to dilute to the scale, shake well, and obtain.

[0084] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions in Table 5 below were respectively adopted:

[0085] Table 5 Screening of different solvents

[0086]

[0087]

[0088] The test results are attached. Figure 3 : The peak shapes of impurities SY012-01A and SY012-01C in two different solvents were consistent, both poor, and the peak shapes could not be improved. Water was still used as the solvent to further explore different gradient programs.

[0089] Example 4

[0090] Solution preparation:

[0091] Mixed solution: prepared as in Example 2.

[0092] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions in Table 6 below were respectively adopted:

[0093] Table 6 Screening of different gradient programs

[0094]

[0095]

[0096] The test results are attached. Figure 4 :The peak shape of impurity SY012-01A in the chromatographic column is normal, the peak shape of impurity SY012-01C is tailing, and the peaks of other impurities can be effectively separated; when the starting proportion of the organic phase is 0%, the peak of impurity SY012-01C is a bulge; as the starting proportion of the organic phase increases, the peak time of impurity SY012-01C peak is advanced; when the starting proportion of the organic phase is 7%, the peak of impurity SY012-01C coincides with the peak of impurity SY012-01; when the starting proportion of the organic phase is 10%, the peak of impurity SY012-01C peaks before the peak of impurity SY012-01, but the separation degree is small. It is tentatively decided to use the gradient program of method 6, and it is planned to replace different types of chromatographic columns for exploration.

[0097] Example 5

[0098] Solution preparation:

[0099] The stock solutions of various impurities were prepared as in Example 2.

[0100] Each impurity location solution: accurately measure 0.5 mL of each impurity stock solution, place it in a 10 mL volumetric flask, dilute to volume with water, shake well, and obtain.

[0101] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions of method 6 in Example 4 were adopted:

[0102] The test results are attached. Figure 5 : The UV absorption spectra of each compound were plotted in the wavelength range of 190-400 nm, and the chromatographic column was optimized using a wavelength of 263 nm.

[0103] Example 6

[0104] Solution preparation:

[0105] Mixed solution: prepared as in Example 2.

[0106] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions in Table 7 below were respectively adopted:

[0107] Table 7 Screening of different chromatographic columns

[0108]

[0109] The test results are attached. Figure 6 : The impurity SY012-01 and the impurity SY012-01C overlap in the mixed solution chromatogram of method 11, and the chromatographic column is not applicable; the separation degree of impurity SY012-01 and impurity SY012-01C in the mixed solution chromatogram of method 12 is slightly poor; the peak shape of impurity SY012-01A in the mixed solution chromatogram of method 13 is slightly tailing; compared with the attached Figure 4 In the chromatogram of method 6, the column COSMOSIL 5-C18, 4.6*250mm, 5μm was tentatively used to further optimize the column temperature.

[0110] Example 7

[0111] Solution preparation:

[0112] Mixed solution: prepared as in Example 2.

[0113] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions in Table 8 below were respectively adopted:

[0114] Table 8 Screening of different column temperatures

[0115]

[0116]

[0117] The test results are attached. Figure 7 : When the column temperature is 20℃ and 30℃, the retention time and peak shape of each impurity in the chromatogram of the mixed solution are basically the same; when the column temperature is 40℃, impurity SY012-01 and impurity SY012-01C overlap in the chromatogram of the mixed solution, and the separation degree of impurity SY012-A and impurity SY012-01D is poor. It is tentatively decided to use a column temperature of 25℃ to further optimize the mobile phase pH.

[0118] Example 8

[0119] Solution preparation:

[0120] Mixed solution: prepared as in Example 2.

[0121] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions in Table 9 below were respectively adopted:

[0122] Table 9 Screening of different mobile phase pH

[0123]

[0124] The test results are attached. Figure 8 :When the mobile phase ApH is 2.5, the separation degree of impurity SY012-01 and impurity SY012-01C in the chromatogram of the mixed solution is poor, and the peaks of impurity SY012-01 and SY012-B overlap; when the mobile phase ApH is 2.8, the chromatographic peak of impurity SY012-01C in the chromatogram of the mixed solution is tailing, and the chromatographic peaks of other impurities can be effectively separated; when the mobile phase ApH is 3.2, the peak order of impurity SY012-A and impurity SY012-01D in the chromatogram of the mixed solution changes; when the mobile phase ApH is 3.5, the peak order of impurity SY012-A and its adjacent impurity peaks in the chromatogram of the mixed solution changes, and the separation degree of impurity SY012-E and the main component peak decreases. Since impurity SY012-01C is the starting material of the starting material, it is planned to explore the detection method of impurity SY012-01C alone, and tentatively use the chromatographic condition of pH 2.8 to study the other 9 impurities.

[0125] Example 9 (Specificity Test)

[0126] Solution preparation:

[0127] Solvent: Water

[0128] Stock solutions of each impurity: Accurately weigh about 16 mg of each of the impurity SY012-01, SY012-01A, SY012-01B, SY012-01D, SY012-A, SY012-B, SY012-C, SY012-D, and SY012-E reference substances, place them in a 20 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0129] Test sample stock solution: Accurately weigh about 4 mg of the test sample and place it in a 10 mL volumetric flask. Add solvent to dissolve and dilute to the scale. Shake well to obtain.

[0130] Stock solution of nikethamide reference substance: Accurately weigh about 16 mg of nikethamide reference substance, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0131] Mixed impurity stock solution: Accurately measure 5 mL of each impurity stock solution, place it in the same 50 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0132] Each impurity location solution: accurately measure 1 mL of each impurity stock solution and place it in a 100 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0133] Nikethamide localizing solution: Accurately measure 1 mL of nikethamide reference stock solution and place it in a 200 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0134] Test solution: Accurately measure 100 μL of nikethamide test stock solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0135] System suitability solution: Accurately measure 1 mL of mixed impurity stock solution and 100 μL of test sample stock solution, place in the same 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0136] Control solution: Accurately measure 20 μL of the nikethamide test solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0137] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were as follows:

[0138] Chromatographic column: Chromatographic column model: COSMOSIL 5-C18-MS-Ⅱ; Chromatographic column filler: octadecyl bonded silica gel; Chromatographic column specifications: inner diameter 4.6mm, length 250mm, filler particle size 5μm;

[0139] Mobile phase: Mobile phase A was 20 mmol potassium dihydrogen phosphate aqueous solution (pH adjusted to 2.8 with phosphoric acid), and mobile phase B was methanol;

[0140] The gradient elution program is as follows:

[0141]

[0142]

[0143] Detection wavelength: 263nm;

[0144] Column temperature: room temperature;

[0145] Flow rate: 1.0 mL / min;

[0146] Injection volume: 10 μL;

[0147] Take 10 μL of each of the blank solvent, system suitability solution, each impurity location solution, test solution and control solution and inject them into the HPLC instrument. Record the chromatogram. The test results are shown in the attached Fig. 9 And Table 10 below.

[0148] Table 10 Specificity test results

[0149]

[0150] From the above results, it can be seen that: the blank solvent has no interference with impurity detection and meets the requirements; the impurities and main components in the system applicability solution can be completely separated, and the separation degree is not less than 2.0; the separation degrees between impurities are greater than 1.0, and the specificity of this method is good.

[0151] Example 10 (Precision Test)

[0152] Solution preparation:

[0153] Solvent: Water

[0154] Each impurity stock solution and nikethamide reference substance stock solution were prepared in the same manner as in Example 9 under the specificity test item.

[0155] Linear stock solution: Accurately measure 2 mL of each impurity stock solution and 1 mL of nikethamide reference stock solution, place them in the same 20 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0156] Mixed impurity reference solution: Accurately measure 1 mL of the linear stock solution and place it in a 10 mL volumetric flask. Add solvent to dilute to the scale and shake well.

[0157] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were the same as those in Example 9 under the specificity test item:

[0158] Take 10 μL of the mixed impurity reference solution and inject it into the high performance liquid chromatograph, record the chromatogram, and the test results are shown in Table 11 below.

[0159] Table 11 Injection precision test results

[0160]

[0161] From the above results, we can know that after 5 consecutive injections of the mixed impurity reference solution, the relative standard deviations of the peak areas of the impurities and main components were 0.09% to 1.78%, all less than 5.0%. This method has good injection precision.

[0162] Example 11 (Linearity and Range Test)

[0163] Solution preparation:

[0164] Solvent: Water

[0165] Linear stock solution: prepared in the same manner as in Example 10 under the precision test item.

[0166] Linear-300% solution: Accurately measure 3 mL of linear stock solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and the solution is ready.

[0167] Linear-200% solution: Accurately measure 2 mL of linear stock solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0168] Linear-150% solution: Accurately measure 3 mL of linear stock solution and place it in a 20 mL volumetric flask, add solvent to dilute to the scale, shake well, and the solution is ready.

[0169] Linear-100% solution: Accurately measure 1 mL of linear stock solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0170] Linear-50% solution: Accurately measure 1 mL of the linear stock solution and place it in a 20 mL volumetric flask. Add solvent to dilute to the scale and shake well.

[0171] Linear-10% solution: Accurately measure 1 mL of the linear stock solution and place it in a 100 mL volumetric flask. Add solvent to dilute to the scale and shake well.

[0172] Linear-5% solution: Accurately measure 1 mL of linear-50% solution and place it in a 10 mL volumetric flask. Add solvent to dilute to the scale and shake well.

[0173] Linear-2% solution: Accurately measure 1 mL of linear-200% solution and place it in a 100 mL volumetric flask. Add solvent to dilute to the scale and shake well.

[0174] Linear-1% solution: Accurately measure 1 mL of linear-10% solution and place it in a 10 mL volumetric flask. Add solvent to dilute to the scale and shake well.

[0175] Linear-0.5% solution: Accurately measure 1 mL of linear-5% solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and the solution is ready.

[0176] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were the same as those in Example 9 under the specificity test item:

[0177] 10 μL of each linear point solution was injected into a high performance liquid chromatograph and the chromatogram was recorded. The test results are shown in Tables 12 to 21 below.

[0178] Table 12 Impurity SY012-01B linearity test results

[0179]

[0180] Table 13 Impurity SY012-01A linear test results

[0181]

[0182]

[0183] Table 14 Impurity SY012-01 linearity test results

[0184]

[0185] Table 15 Impurity SY012-B linearity test results

[0186]

[0187] Table 16 Impurity SY012-A linearity test results

[0188]

[0189] Table 17 Impurity SY012-01D linear test results

[0190]

[0191] Table 18 Impurity SY012-C linearity test results

[0192]

[0193]

[0194] Table 19 Impurity SY012-E linearity test results

[0195]

[0196] Table 20 Nikethamide linearity test results

[0197]

[0198] Table 21 Impurity SY012-D linear test results

[0199]

[0200]

[0201] From the above results, it can be seen that the impurity SY012-01B has a good linear relationship in the concentration range of 0.04048 to 24.29 μg / mL; the impurity SY012-01A has a good linear relationship in the concentration range of 0.04132 to 24.79 μg / mL; the impurity SY012-01 has a good linear relationship in the concentration range of 0.04127 to 24.76 μg / mL; the impurity SY012-B has a good linear relationship in the concentration range of 0.04066 to 24.40 μg / mL; the impurity SY012-A has a good linear relationship in the concentration range of 0.08549 to 25.65 μg / mL The linear relationship of impurity SY012-01D is good in the concentration range of 0.08155-24.47μg / mL; the linear relationship of impurity SY012-C is good in the concentration range of 0.04063-24.38μg / mL; the linear relationship of impurity SY012-E is good in the concentration range of 0.09770-29.31μg / mL; the linear relationship of nikethamide is good in the concentration range of 0.1687-25.31μg / mL; the linear relationship of impurity SY012-D is good in the concentration range of 0.08177-24.53μg / mL. The linear correlation coefficient R of each known impurity and nikethamide is greater than 0.99, which meets the requirements.

[0202] Example 12 (Detection limit test)

[0203] Solution preparation:

[0204] Solvent: Water

[0205] The linear point solutions prepared under the linear range test item of Example 11 were diluted step by step, and the solution with a signal-to-noise ratio of not less than 10:1 was used as the quantitative limit solution, and the solution with a signal-to-noise ratio of not less than 3:1 was used as the detection limit solution.

[0206] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were the same as those in Example 9 under the specificity test item:

[0207] 10 μL of each linear point solution was injected into the high performance liquid chromatograph and the chromatogram was recorded. The test results are shown in Table 22 below.

[0208] Table 22 Detection limit results

[0209]

[0210]

[0211] From the above results, it can be seen that the detection limit concentration of the impurity SY012-01B detected by this method is 0.01619μg / mL, which is equivalent to 0.20% of the limit concentration; the quantitative limit concentration is 0.04048μg / mL, which is equivalent to 0.51% of the limit concentration; the detection limit concentration of the impurity SY012-01A is 0.01653μg / mL, which is equivalent to 0.21% of the limit concentration; the quantitative limit concentration is 0.04132μg / mL, which is equivalent to 0.52% of the limit concentration; the detection limit concentration of the impurity SY012-01 is 0.01651 ... g / mL, equivalent to 0.21% of the limit concentration; the quantitative limit concentration is 0.04127μg / mL, equivalent to 0.52% of the limit concentration; the detection limit concentration of impurity SY012-B is 0.01626μg / mL, equivalent to 0.20% of the limit concentration; the quantitative limit concentration is 0.04066μg / mL, equivalent to 0.51% of the limit concentration; the detection limit concentration of impurity SY012-A is 0.04275μg / mL, equivalent to 0.53% of the limit concentration; the quantitative limit concentration is 0.08549μg / mL, equivalent to the limit concentration The detection limit of impurity SY012-01D is 0.04078μg / mL, equivalent to 0.51% of the limit concentration; the quantitative limit is 0.08155μg / mL, equivalent to 1.0% of the limit concentration; the detection limit of impurity SY012-C is 0.008127μg / mL, equivalent to 0.10% of the limit concentration; the quantitative limit is 0.04063μg / mL, equivalent to 0.51% of the limit concentration; the detection limit of impurity SY012-E is 0.01954μg / mL, equivalent to The detection limit of SY012-D was 0.04089 μg / mL, equivalent to 0.51% of the limit concentration; the limit of quantification was 0.08177 μg / mL, equivalent to 1.0% of the limit concentration. The detection limit of the main component was 0.08435 μg / mL, equivalent to 1.1% of the limit concentration; the limit of quantification was 0.1687 μg / mL, equivalent to 2.1% of the limit concentration; the detection limit of SY012-D was 0.04089 μg / mL, equivalent to 0.51% of the limit concentration; the limit of quantification was 0.08177 μg / mL, equivalent to 1.0% of the limit concentration. This method has high detection sensitivity.

[0212] Example 13 (Repeatability test)

[0213] Solution preparation:

[0214] Solvent: Water

[0215] Test solution: Take an appropriate amount of nikethamide and dilute it with solvent to make a solution containing about 4 mg per 1 mL. Prepare 6 copies in parallel.

[0216] Control solution: Accurately measure an appropriate amount of the test sample solution, add solvent and quantitatively dilute it to make a solution containing about 8 μg per 1 mL.

[0217] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were the same as those in Example 9 under the specificity test item:

[0218] 10 μL of the test solution and control solution were respectively injected into the high performance liquid chromatograph. The test results are shown in Table 23 below.

[0219] Table 23 Repeatability test results

[0220]

[0221] From the above results, we can see that the difference between a single impurity and the mean in the 6 test solutions is in the range of 0-0.01%, which is no more than 20% of the limit; the difference between the sum of the impurities and the mean is in the range of 0-0.01%, which is no more than 10% of the limit. This method has good repeatability.

[0222] Example 14 (Accuracy Test)

[0223] Solution preparation:

[0224] Solvent: Water

[0225] Mixed impurity stock solution: prepared in the same manner as in Example 9 under the specificity test item.

[0226] Stock solution of test sample: Take an appropriate amount of nikethamide and dilute it with solvent to make a solution containing about 40 mg per 1 mL.

[0227] 50% recovery solution: Accurately measure 1 mL of the test sample stock solution and the mixed impurity stock solution, place them in the same 20 mL volumetric flask, add solvent to dilute to the scale, shake well, and use this as the 50% recovery solution. Prepare 3 copies in parallel.

[0228] 100% recovery solution: Accurately measure 1 mL each of the test sample stock solution and the mixed impurity stock solution, place them in the same 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and use this as the 100% recovery solution. Prepare 3 copies in parallel.

[0229] 150% recovery solution: Accurately measure 3 mL of the test sample stock solution and the mixed impurity stock solution, place them in the same 20 mL volumetric flask, add solvent to dilute to the scale, shake well, and use this as the 150% recovery solution. Prepare 3 copies in parallel.

[0230] 50% recovery solution - self-control solution: Accurately measure 20 μL of 50% recovery solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0231] 100% recovery solution - self-control solution: Accurately measure 20 μL of 100% recovery solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0232] 150% recovery solution - self-control solution: Accurately measure 20 μL of 150% recovery solution and place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0233] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were the same as those in Example 9 under the specificity test item:

[0234] 10 μL of each of the above 50%, 100%, 150% recovery solutions and their own control solutions were injected into a high performance liquid chromatograph, and the chromatograms were recorded. The test results are shown in Tables 24 to 32 below.

[0235] Table 24 Impurity SY012-01B accuracy test results

[0236]

[0237]

[0238] Table 25 Impurity SY012-01A accuracy test results

[0239]

[0240] Table 26 Impurity SY012-01 accuracy test results

[0241]

[0242] Table 27 Impurity SY012-B accuracy test results

[0243]

[0244]

[0245] Table 28 Impurity SY012-A accuracy test results

[0246]

[0247] Table 29 Impurity SY012-01D accuracy test results

[0248]

[0249] Table 30 Impurity SY012-C Accuracy Test Results

[0250]

[0251]

[0252] Table 31 Impurity SY012-E accuracy test results

[0253]

[0254] Table 32 Impurity SY012-D accuracy test results

[0255]

[0256] From the above results, it can be seen that the average recovery rate of each impurity is in the range of 92.34% to 99.86%, which meets the requirements and the method has good accuracy.

[0257] Example 15 (Solution Stability Test)

[0258] Solution preparation:

[0259] Solvent: Water

[0260] Test solution, system suitability solution and control solution: prepared as in Example 9 under the specificity test item, and placed at room temperature and 2-8°C respectively.

[0261] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were the same as those in Example 9 under the specificity test item:

[0262] Take 10 μL of the above test solution and its own control solution and inject them into the high performance liquid chromatograph, record the chromatogram, and the test results are shown below. Fig.10 And Table 33 below.

[0263] Table 33 Test results of the stability of the test solution

[0264]

[0265] From the above results, it can be seen that: the impurity peaks in the system suitability solution are clearly visible; when the test solution is placed at room temperature and 2-8℃ low temperature conditions for 6 days, the difference between a single impurity and 0 day is no more than 20% of the limit, and the difference between the sum of the impurities and 0 day does not exceed 10% of the limit. The test solution is stable for at least 6 days at room temperature and 2-8℃ low temperature conditions.

[0266] Example 16 (Durability Test)

[0267] Solution preparation:

[0268] Mixed impurity reference solution, system suitability solution, test solution and its own reference solution: prepared as in Example 9 under the specificity test item, all placed at room temperature and 2-8°C.

[0269] Reverse phase high performance liquid chromatography was used for detection, and the detection conditions were the same as those in Example 9 under the specificity test item:

[0270] Take 10 μL of each of the above-mentioned mixed impurity reference solution, system suitability solution, test solution and its own control solution and inject them into the high performance liquid chromatograph, record the chromatogram, and the test results are shown in Tables 34 to 38 below.

[0271] Table 34 Durability test separation results (system suitability solution)

[0272]

[0273]

[0274] Table 35 Durability correction factor test results (mixed impurity reference solution)

[0275]

[0276] Table 36 Durability relative retention time test results (system suitability solution)

[0277]

[0278]

[0279] Table 37 Test results of durability and relative retention time ratio under standard conditions (system suitability solution)

[0280]

[0281] Table 38 Durability related substance test results (test solution)

[0282]

[0283]

[0284] Note: Chromatographic column 1: YMC-Triart C18, 4.6*150mm, 5μm

[0285] Column 2: phenomenex C18, 4.6*150mm, 5μm

[0286] Column 3: Thermo Hypersil GOLD, 4.6*250mm, 5μm

[0287] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A method for determining related substances of nikethamide, characterized in that: Reverse phase high performance liquid chromatography was used, with octadecyl bonded silica gel as the filler of the chromatographic column, 20 mmol potassium dihydrogen phosphate aqueous solution as the mobile phase A, and methanol as the mobile phase B, to separate nikethamide and related impurities by gradient elution; The mobile phase A is adjusted to a pH value of 2.8 with phosphoric acid; The relevant impurities are one or more of impurities SY012-01, SY012-01A, SY012-01B, SY012-01C, SY012-01D, SY012-A, SY012-B, SY012-C, SY012-D, and SY012-E; The specific structural formula of the relevant impurities is as follows:

2. The method for determining the related substances of nikethamide according to claim 1, characterized in that: The gradient elution procedure is as follows:

3. The method for determining the related substances of nikethamide according to claim 1, characterized in that: The chromatographic column is one of COSMOSIL C18.

4. The method for determining the related substances of nikethamide according to claim 3, characterized in that: The inner diameter of the chromatographic column is 4.6 mm, the length is 250 mm, and the filler particle size is 5 μm.

5. The method for determining the related substances of nikethamide according to claim 1, characterized in that: The column temperature of the chromatographic column is 20-30°C, preferably 25°C.

6. The method for determining the related substances of nikethamide according to claim 1, characterized in that: The injection volume of the gradient elution is 10 μL to 50 μL, preferably 10 μL.

7. The method for determining the related substances of nikethamide according to claim 1, characterized in that: The flow rate of the gradient elution is 0.8-1.2 mL / min, preferably 1.0 mL / min.

8. The method for determining the related substances of nikethamide according to claim 1, characterized in that: Use ultraviolet light detection with a wavelength of 262-265 nm, preferably 263 nm.

9. The method for determining the related substances of nikethamide according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) Prepare the test solution: take the product and dilute it with water to make a solution containing about 4 μg of nikethamide per 1 mL; prepare the control solution: accurately measure an appropriate amount of the test solution and quantitatively dilute it with water to make a solution containing about 8 μg of nikethamide per 1 mL; (2) Separation: Reverse phase high performance liquid chromatography was used, with a chromatographic column using octadecyl bonded silica as filler, a 20 mmol potassium dihydrogen phosphate aqueous solution as mobile phase A, methanol as mobile phase B, and mobile phase A adjusted to pH 2.8 with phosphoric acid, and the test solution and control solution obtained in step (1) were subjected to gradient elution. (3) Detection: Use ultraviolet light with a wavelength of 262 to 265 nm, preferably 263 nm.

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