A quality control method for Vibergron

Abstract

The present invention relates to the technical field of drug analysis, and specifically to a quality control method for Vibergolone, which is determined by conventional high performance liquid chromatography: the chromatographic column is a C18 column; the detection wavelength is 250nm; gradient elution is adopted; potassium hydrogen phosphate solution and methanol are used as mobile phase A, and acetonitrile is used as mobile phase B; the flow rate is 1.0ml / min; the column temperature is 40°C; the injection volume is 5μL; and the elution time is 45min. Verification shows that the method has the advantages of simple operation, strong selectivity, high sensitivity, and good accuracy, can effectively elute and separate related impurities in Vibergolone raw materials, and is suitable for the effective determination of Vibergolone related substances. This is a simple, efficient and easy-to-implement analytical method, which can be used for the determination of its effective content at the same time.

Description

Technical Field

[0001] The invention relates to a quality control method of vibergron, belonging to the technical field of drug analysis. Background Art

[0002] Vibegron is a beta-3 adrenergic agonist used to treat urge incontinence, urgency and frequency of urination in patients with overactive bladder (OAB, first approved indication). Other indications under development include prostatic hyperplasia, neurogenic detrusor overactivity, etc. Non-investigational indications include endocrine system diseases, hypertension, irritable bowel syndrome and pain. It has broad market application prospects.

[0003] The structural formula of Vibegron (No. VSD23003) is: .

[0004] The synthetic route of Vibegron (No. VSD23003) is:

[0005] .

[0006] From the synthesis route, it can be seen that the substances involved in the preparation process of Vibergron include:

[0007] (1) VSD23003-Q1 ;

[0008] (2) VSD23003-M1 ;

[0009] (3) VSD23003-M4 ;

[0010] (4) VSD23003-M5 (impurity A) ;

[0011] (5) Impurity B ;

[0012] (6) Impurity E ;

[0013] (7) Impurity F ;

[0014] (8) VSD23003 .

[0015] It can be seen from the above compound structures that vibergolone has multiple chiral centers. The physical and chemical properties of the isomer (impurity B) and the vibergolone molecule are slightly different, and there is no obvious difference in the retention behavior on the chromatographic column, which makes separation difficult. The polarity of impurity E and impurity VSD23003-M1 is quite different from that of the vibergolone molecule, and the retention behavior on the chromatographic column is quite different, which makes elution difficult. How to simultaneously and effectively control the content of all related substances has become a difficult problem in the quality control of vibergolone.

[0016] CN113816917B discloses a method for preparing a Vibergolone intermediate. The patent reports a compound The purity test method of impurity F was used to test the purity of Vibergolone API. However, the test confirmed that this method is not suitable for the detection of related substances of Vibergolone API, mainly in the following aspects: (1) Vibergolone and its adjacent impurity peaks cannot be effectively separated; (2) Impurity E is not retained under this method; (3) VSD23003-M1 is too strongly retained to be effectively eluted, resulting in incomplete impurity detection; (4) The column temperature is 10°C, which is not conducive to the implementation of some instruments without column temperature control or column oven without active cooling function.

[0017] CN117434179A discloses a purity detection method for mirabegron raw materials. Mirabegron and vibergron belong to the same class of beron compounds, and their structures have certain similarities, so their analysis methods have certain reference significance. However, it has been confirmed by experiments that under the conditions of the disclosed method, the peaks of vibergron and its diastereoisomer (impurity B) completely overlap and cannot be separated, so it is not suitable for the detection of related substances of vibergron raw materials.

[0018] At present, there is no public report on the quality control method of vibergolone raw materials. In order to effectively control its quality to ensure its clinical safety and effectiveness, it is urgent to develop an analytical method for the determination of vibergolone-related substances and their contents. Summary of the invention

[0019] The present invention aims at the deficiencies in the prior art and provides a quality control method for Vibergolone, which can separate and detect related substances involved in Vibergolone raw material medicine and can also be used for determining the effective content thereof.

[0020] The technical solution of the present invention to solve the above technical problems is as follows: A quality control method of Vibergolone, using high performance liquid chromatography to simultaneously perform quantitative analysis on substances in Vibergolone raw materials, comprising the following steps:

[0021] S1. Solution preparation

[0022] Preparation of blank solvent: methylene diphosphonic acid aqueous solution and methanol are mixed evenly to obtain blank solvent;

[0023] Preparation of test solution: Accurately weigh the Vibegron API to be tested, dissolve it in blank solvent and dilute it to make a solution containing 2.0 mg of the test solution per 1 mL;

[0024] Preparation of control solution: Accurately measure the test solution and dilute it with blank solvent to obtain a control solution with a concentration of 2 μg / mL;

[0025] Preparation of reference solution: accurately weigh the vibergolone reference substance, dissolve it in blank solvent and dilute it to make a reference solution containing 2.0 mg of vibergolone reference substance per 1 mL;

[0026] Preparation of impurity reference substance stock solutions: Accurately weigh impurity A reference substance, impurity B reference substance, impurity E reference substance, impurity F reference substance, VSD23003-Q1, VSD23003-M1, and VSD23003-M4 reference substances, respectively, dissolve and dilute with blank solvent to prepare 0.02 mg of each impurity reference substance stock solution per 1 mL, and then gradually dilute to the required concentration;

[0027] Among them, VSD23003-Q1 is ; VSD23003-M1 is ; VSD23003-M4 is ; Impurity A is ; Impurity B is ; Impurity E is ; Impurity F is ;

[0028] S2. Quantitative analysis of substances in the Vibegron API was performed simultaneously using high performance liquid chromatography:

[0029] The detector of the high performance liquid chromatograph is an ultraviolet detector, and the chromatographic conditions are as follows: the mobile phase is phosphate aqueous solution and methanol as mobile phase A, and acetonitrile as mobile phase B; a gradient elution method is adopted; the flow rate of the mobile phase is 0.8mL / min~1.2mL / min; the chromatographic column is a C18 or C8 column; the column temperature is 30°C~45°C; the injection volume is 4μL~6μL; and the elution time is 45min.

[0030] Furthermore, in step S1, the volume ratio of the methylene diphosphonic acid aqueous solution to methanol in the blank solvent is 1:(0.99-1.01), and the concentration of the methylene diphosphonic acid aqueous solution is 9.9-10.1 mmol / L.

[0031] Preferably, in step S1, the volume ratio of the methylene diphosphonic acid aqueous solution to methanol in the blank solvent is 1:1, and the concentration of the methylene diphosphonic acid aqueous solution is 10 mmol / L.

[0032] Furthermore, in step S2, in the mobile phase A, the volume ratio of the phosphate aqueous solution to methanol is 95:5, and the concentration of phosphate in the phosphate aqueous solution is 5 mmol / L to 20 mmol / L.

[0033] Furthermore, in step S2, the phosphate in the phosphate aqueous solution is dipotassium hydrogen phosphate, ammonium phosphate, sodium phosphate, ammonium formate or ammonium acetate.

[0034] Preferably, in step S2, the mobile phase A consists of a dipotassium hydrogen phosphate aqueous solution and methanol, the volume ratio of the dipotassium hydrogen phosphate aqueous solution to methanol is 95:5, and the concentration of the dipotassium hydrogen phosphate aqueous solution is 10 mmol / L.

[0035] Preferably, in step S2, the chromatographic column is a 4.6 mm×250 mm, 5 μm C18 chromatographic column.

[0036] Preferably, in step S2, the flow rate of the mobile phase is 1.0 mL / min.

[0037] Preferably, in step S2, the injection volume is 5 μL.

[0038] Preferably, in step S2, the column temperature is 40°C.

[0039] Preferably, in step S2, the gradient elution is performed according to the following linear gradient:

[0040] 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%;

[0041] 2) When the elution time is 3 min, the volume fraction of mobile phase A is 100%;

[0042] 3) When the elution time is 5 min, the volume fraction of mobile phase A is 78%, and the volume fraction of mobile phase B is 22%;

[0043] 4) When the elution time is 21 min, the volume fraction of mobile phase A is 65%, and the volume fraction of mobile phase B is 35%;

[0044] 5) When the elution time is 27 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%;

[0045] 6) When the elution time is 39 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%;

[0046] 7) When the elution time is 39.1 min, the volume fraction of mobile phase A is 100%;

[0047] 8) When the elution time is 45 minutes, the volume fraction of mobile phase A is 100%.

[0048] The beneficial effects of the present invention are:

[0049] (1) The present invention can effectively separate various impurities in the Vibergol API, especially isomeric impurities with similar physical and chemical properties (difficult to separate) and related impurities with large physical and chemical properties (large difference in retention factors, difficult to elute), and the blank solvent does not interfere with the detection process. The method has strong specificity and good selectivity.

[0050] (2) The solvent of the present invention can avoid the problem of poor impurity peak shape caused by solvent effect. At the same time, methylene diphosphonic acid has the function of masking metal ions (introduced by metal catalysts used in the process) to reduce the damage of metal ions to the performance of the chromatographic column.

[0051] (3) The present invention is also applicable to the process control of multiple intermediate products of Vibergron, and has a wide range of applications, thereby making it more convenient and effective to study the impurity spectrum in the synthesis process and effectively control the product quality.

[0052] (4) The present invention can achieve simultaneous determination of product-related substances and their contents, effectively reducing the testing cost and simplifying the verification of the analytical method and the subsequent testing process of the production and testing department.

[0053] (5) The present invention adopts conventional high performance liquid chromatography, which does not require complicated pretreatment, has low cost, strong universality, high precision, good reproducibility, has a strong stability indication function, and can be smoothly transferred to different laboratories for quality control of the Vibergolone API, thereby ensuring the safety, effectiveness and quality controllability of the Vibergolone API. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is the liquid chromatogram of the blank solvent in Example 1;

[0055] Figure 2 It is the liquid chromatogram of the reference substance solution in Example 1;

[0056] Figure 3 It is the liquid chromatogram of the test solution in Example 1;

[0057] Figure 4 The liquid chromatogram of the spiked test solution in Example 1;

[0058] Figure 5 The liquid chromatogram of the spiked test solution in Example 2;

[0059] Figure 6 The liquid chromatogram of the spiked test solution in Example 3;

[0060] Figure 7The liquid chromatogram of the spiked test solution in Example 4;

[0061] Figure 8 It is the liquid chromatogram of the blank solvent in Comparative Example 1;

[0062] Fig. 9 It is the liquid chromatogram of the test solution in Comparative Example 1;

[0063] Fig.10 This is the liquid chromatogram of the spiked test solution in Comparative Example 1;

[0064] Fig.11 It is the liquid chromatogram of the blank solvent in Comparative Example 2;

[0065] Fig.12 It is the liquid chromatogram of the test solution in Comparative Example 2. DETAILED DESCRIPTION

[0066] The specific implementation of the present invention is described in detail below. The present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used are only for describing specific embodiments and are not intended to limit the present invention.

[0068] A quality control method for Vibergolone, which uses high performance liquid chromatography to simultaneously perform quantitative analysis on substances in Vibergolone API (involving key materials, intermediates and multiple related impurities and main components generated in the process thereof), comprises the following steps:

[0069] S1. Solution preparation

[0070] Preparation of blank solvent: methylene diphosphonic acid aqueous solution and methanol are mixed evenly to obtain blank solvent;

[0071] Preparation of test solution: Accurately weigh an appropriate amount of the Vibegron API to be tested, dissolve it in blank solvent and dilute it to make a test solution containing 2.0 mg per 1 mL;

[0072] 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 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 solvent, and shake well to obtain a control solution with a concentration of 2 μg / mL;

[0073] Preparation of reference solution: accurately weigh an appropriate amount of Vibergolon reference substance, dissolve it in blank solvent and dilute it to make a reference solution containing 2.0 mg of Vibergolon reference substance per 1 mL;

[0074] Preparation of impurity reference substance stock solutions: Accurately weigh appropriate amounts of impurity A reference substance, impurity B reference substance, impurity E reference substance, impurity F reference substance, VSD23003-Q1, VSD23003-M1, and VSD23003-M4 reference substances, respectively, dissolve and dilute with blank solvent to prepare impurity reference substance stock solutions containing 0.02 mg of impurity reference substance per 1 mL, and then gradually dilute to the required concentration;

[0075] S2. Use high performance liquid chromatography to simultaneously perform quantitative analysis on the substances in the Vibergolone API: inject the test solution, control solution, reference substance solution, and impurity reference substance stock solution into the high performance liquid chromatography for detection.

[0076] The detector of the high performance liquid chromatograph is an ultraviolet detector, and the chromatographic conditions are as follows: the mobile phase is phosphate aqueous solution and methanol as mobile phase A, and acetonitrile as mobile phase B; a gradient elution method is adopted; the flow rate of the mobile phase is 0.8mL / min~1.2mL / min; the chromatographic column is a C18 or C8 column; the column temperature is 30°C~45°C; the injection volume is 4μL~6μL; and the elution time is 45min.

[0077] Specifically, in step S1, the volume ratio of the methylene diphosphonic acid aqueous solution to methanol in the blank solvent is 1:(0.99-1.01), and the concentration of the methylene diphosphonic acid aqueous solution is 9.9-10.1 mmol / L.

[0078] Preferably, in step S1, the volume ratio of the methylene diphosphonic acid aqueous solution to methanol in the blank solvent is 1:1, and the concentration of the methylene diphosphonic acid aqueous solution is 10 mmol / L.

[0079] Specifically, in step S2, in the mobile phase A, the volume ratio of the phosphate aqueous solution to methanol is 95:5, and the concentration of phosphate in the phosphate aqueous solution is 5 mmol / L to 20 mmol / L;

[0080] Specifically, in step S2, the phosphate in the phosphate aqueous solution is dipotassium hydrogen phosphate, ammonium phosphate, sodium phosphate, ammonium formate or ammonium acetate.

[0081] Preferably, in step S2, the mobile phase A consists of a dipotassium hydrogen phosphate aqueous solution and methanol, the volume ratio of the dipotassium hydrogen phosphate aqueous solution to methanol is 95:5, and the concentration of the dipotassium hydrogen phosphate aqueous solution is 10 mmol / L.

[0082] Preferably, in step S2, the chromatographic column is a 4.6 mm×250 mm, 5 μm C18 chromatographic column.

[0083] Preferably, in step S2, the flow rate of the mobile phase is 1.0 mL / min.

[0084] Preferably, in step S2, the injection volume is 5 μL.

[0085] Preferably, in step S2, the column temperature is 40°C.

[0086] Preferably, in step S2, the gradient elution is performed according to the following linear gradient:

[0087] 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%;

[0088] 2) When the elution time is 3 min, the volume fraction of mobile phase A is 100%;

[0089] 3) When the elution time is 5 min, the volume fraction of mobile phase A is 78%, and the volume fraction of mobile phase B is 22%;

[0090] 4) When the elution time is 21 min, the volume fraction of mobile phase A is 65%, and the volume fraction of mobile phase B is 35%;

[0091] 5) When the elution time is 27 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%;

[0092] 6) When the elution time is 39 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%;

[0093] 7) When the elution time is 39.1 min, the volume fraction of mobile phase A is 100%;

[0094] 8) When the elution time is 45 minutes, the volume fraction of mobile phase A is 100%.

[0095] The specific test procedures are:

[0096] Accurately measure 5 μL of the control solution, test solution, and reference solution in step S1, respectively, and inject them into the liquid chromatograph to record the chromatogram. Calculate the impurity content by the main component self-reference method with the correction factor, and calculate the vibergron content by the peak area according to the external standard method. The calculation formulas are:

[0097] Impurity content (%) = relative correction factor × A S0 / Ar×0.1%;

[0098] Where:

[0099] AS0 is: the peak area of ​​each impurity in the test solution;

[0100] Ar is: the main peak area of ​​the control solution.

[0101] ;

[0102] Where:

[0103] As is: the peak area of ​​vibergron in the reference solution;

[0104] M S is: the amount of Vibergron reference substance weighed when preparing the reference substance solution (mg);

[0105] A T is: the peak area of ​​vibergron in the test solution;

[0106] M T is: the mass of the Vibegron raw material to be tested weighed when preparing the test solution (mg);

[0107] ρ is: the content of vibergolone reference substance (%).

[0108] Among them, relative correction factor = slope of the principal component linear equation / slope of the impurity linear equation.

[0109] 1. Detection Example

[0110] Example 1

[0111] (1) Solution preparation

[0112] Blank solvent: a mixture of 10 mmol / L methylene diphosphonic acid aqueous solution and methanol (volume ratio 1:1);

[0113] Reference solution: Take an appropriate amount of Vibegron reference substance, dissolve it in blank solvent and dilute it to make a solution containing about 2.0 mg per 1 mL as the reference solution.

[0114] Test solution: Take an appropriate amount of the Vibegron API to be tested, dissolve it in a solvent and dilute it to make a solution containing about 2.0 mg per 1 mL, which is used as the test solution.

[0115] Preparation of control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add 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 solvent to dilute to the scale, shake well to obtain a control solution with a concentration of 2 μg / mL.

[0116] Spiked test solution: Take appropriate amount of impurity A reference substance, impurity B reference substance, impurity E reference substance, impurity F reference substance, VSD23003-Q1, VSD23003-M1, VSD23003-M4 reference substance, and vibergron reference substance, dissolve in blank solvent and quantitatively dilute to make a solution containing approximately 2.0 mg of vibergron per 1 mL and approximately 2 ug of other impurities.

[0117] (2) HPLC was used to simultaneously conduct quantitative analysis of substances in the vibergolone bulk drug substance: blank solvent, test solution, reference solution, reference substance solution, impurity reference substance stock solution, and spiked test solution were injected into the HPLC for detection.

[0118] Liquid chromatography conditions: The chromatographic column is Welch Xtimate C18 (4.6×250mm, 5μm); 10mmol / L potassium dihydrogen phosphate aqueous solution-methanol (volume ratio of 95:5) solution is used as mobile phase A, and acetonitrile solution is used as mobile phase B; the detection wavelength is 250nm; the flow rate is 1.0mL / min; the column temperature is 40℃; the injection volume is 5μL; the elution time is 45min; the detector is a UV detector.

[0119] The elution gradient process is:

[0120] 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%;

[0121] 2) When the elution time is 3 min, the volume fraction of mobile phase A is 100%;

[0122] 3) When the elution time is 5 min, the volume fraction of mobile phase A is 78%, and the volume fraction of mobile phase B is 22%;

[0123] 4) When the elution time is 21 min, the volume fraction of mobile phase A is 65%, and the volume fraction of mobile phase B is 35%;

[0124] 5) When the elution time is 27 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%;

[0125] 6) When the elution time is 39 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%;

[0126] 7) When the elution time is 39.1 min, the volume fraction of mobile phase A is 100%;

[0127] 8) When the elution time is 45 minutes, the volume fraction of mobile phase A is 100%.

[0128] The chromatograms of the blank solvent, reference solution, test solution and spiked test solution in this example are shown in Figure 2. Figure 1-Figure 4 ;from Figure 1-Figure 4 It can be seen that the separation between Vibergron and each impurity meets the requirements. The separation between unknown impurities and known impurities, and between known impurities and known impurities is good, and the method has good specificity.

[0129] Example 2

[0130] The difference between this embodiment 2 and embodiment 1 is that the chromatographic column is changed.

[0131] (1) Solution preparation

[0132] The solution preparation is the same as that of Example 1.

[0133] (2) HPLC was used to simultaneously conduct quantitative analysis of substances in the vibergolone bulk drug substance: blank solvent, test solution, reference solution, reference substance solution, impurity reference substance stock solution, and spiked test solution were injected into the HPLC for detection.

[0134] Liquid chromatography conditions: chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 10mmol / L potassium dihydrogen phosphate-methanol (95:5) solution as mobile phase A, acetonitrile solution as mobile phase B; detection wavelength: 250nm; flow rate: 1.0mL / min; column temperature: 40°C; injection volume: 5μL; elution time: 45min; detector: UV detector. The elution gradient process is the same as that of Example 1.

[0135] In the liquid chromatogram of Example 2, the main peak of Vibergron and the impurities as well as the impurities were well separated, and the sensitivity was slightly lower than that of Example 1. Figure 5 .

[0136] Example 3

[0137] The difference between Example 3 and Example 1 is that the column temperature is changed.

[0138] (1) Solution preparation

[0139] The solution preparation is the same as that of Example 1.

[0140] (2) HPLC was used to simultaneously conduct quantitative analysis of substances in the vibergolone bulk drug substance: blank solvent, test solution, reference solution, reference substance solution, impurity reference substance stock solution, and spiked test solution were injected into the HPLC for detection.

[0141] Liquid chromatography conditions: the chromatographic column is Welch Xtimate C18 (4.6×250mm, 5μm); 10mmol / L potassium dihydrogen phosphate-methanol (95:5) solution is used as mobile phase A, and acetonitrile solution is used as mobile phase B; the detection wavelength is 250nm; the flow rate is 1.0mL / min; the column temperature is 35°C; the injection volume is 5μL; the elution time is 45min; the detector is an ultraviolet detector, and the elution gradient is the same as the gradient in Example 1.

[0142] In the liquid chromatogram of Example 3, the main peak of Vibergron and the impurities as well as the impurities were well separated, see Figure 6 .

[0143] Example 4

[0144] The difference between this embodiment 4 and embodiment 1 is that the flow rate is changed.

[0145] (1) Solution preparation

[0146] The solution preparation is the same as that of Example 1.

[0147] (2) HPLC was used to simultaneously conduct quantitative analysis of substances in the vibergolone bulk drug substance: blank solvent, test solution, reference solution, reference substance solution, impurity reference substance stock solution, and spiked test solution were injected into the HPLC for detection.

[0148] Liquid chromatography conditions: the chromatographic column is Welch Xtimate C18 (4.6×250mm, 5μm); 10mmol / L potassium dihydrogen phosphate-methanol (95:5) solution is used as mobile phase A, and acetonitrile solution is used as mobile phase B; the detection wavelength is 250nm; the flow rate is 0.8mL / min; the column temperature is 40°C; the injection volume is 5μL; the elution time is 45min; the detector is an ultraviolet detector, and the elution gradient is the same as the gradient in Example 1.

[0149] In the liquid chromatogram of Example 4, the main peak of Vibergron and the impurities as well as the impurities were well separated, see Figure 7 .

[0150] Comparative Example 1

[0151] The detection of Vibergron was performed by one of the conventional methods.

[0152] (1) Solution preparation

[0153] Blank solvent: 10mmol / L EDTA aqueous solution and acetonitrile (volume ratio of 1:1) were mixed evenly to obtain the blank solvent.

[0154] Test solution: the same as the test solution in Example 1, except that the blank solvent described in this comparative example 1 is used to prepare the test solution;

[0155] Spiked test solution: The spiked test solution is the same as the spiked test solution in Example 1, except that the blank solvent described in this comparative example 1 is used to prepare the spiked test solution.

[0156] (2) Inject blank solvent, test solution, and spiked test solution into HPLC for detection.

[0157] Liquid chromatography conditions: Chromatographic column: XBridge C18 (4.6×250mm, 5μm); 10mmol / L ammonium formate buffer (add 10mmol ammonium formate and 5mL ammonia water to every 1000mL water) as mobile phase A, acetonitrile solution as mobile phase B; detection wavelength is 236nm; flow rate is 1.0mL / min; column temperature is 10℃; injection volume is 5μL; elution time is 42min; detector is ultraviolet detector.

[0158] The elution gradient process is:

[0159] 1) At elution time 0 min, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%;

[0160] 2) When the elution time is 5 min, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%;

[0161] 3) When the elution time is 15 min, the volume fraction of mobile phase A is 67% and the volume fraction of mobile phase B is 33%;

[0162] 4) When the elution time is 25 min, the volume fraction of mobile phase A is 67%, and the volume fraction of mobile phase B is 33%;

[0163] 5) When the elution time is 30 min, the volume fraction of mobile phase A is 10% and the volume fraction of mobile phase B is 90%;

[0164] 6) When the elution time is 35 min, the volume fraction of mobile phase A is 10%, and the volume fraction of mobile phase B is 90%;

[0165] 7) When the elution time is 36 min, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%;

[0166] 8) When the elution time is 42 minutes, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%.

[0167] Under this method, the main peak of Vibergolone is tailing, and the separation degree of impurities after the main peak does not meet the requirements. Impurity E is not retained and cannot be determined (peaks are produced at the solvent peak and there is interference). VSD23003-M1 cannot be detected due to too strong retention. It is not suitable for purity testing of Vibergolone API, see Figure 8~Figure 10 , and the 10mmol / L EDTA aqueous solution used in this method is one of the components of the blank solvent. EDTA can only be slightly soluble in the aqueous solution and cannot be completely dissolved.

[0168] Comparative Example 2

[0169] Another conventional method was used for the detection of Vibergron.

[0170] (1) Solution preparation

[0171] Blank solvent: 15% methanol in water

[0172] Test solution: The same as the test solution in Example 1, except that the blank solvent described in Comparative Example 2 is used to prepare the test solution.

[0173] (2) Inject the blank solvent and the test solution into the HPLC for detection.

[0174] Liquid chromatography conditions:

[0175] Ultimate AQ- C18 (4.6×250mm, 5μm); phosphate buffer (containing 0.03 mol potassium hexafluorophosphate and 0.03 mol potassium dihydrogen phosphate per 1000 ml of water, and the pH value was adjusted to 6.0 with phosphoric acid) as mobile phase A, methanol:acetonitrile = 50:50 (volume ratio) solution as mobile phase B; detection wavelength is 250nm; flow rate is 1.0mL / min; column temperature is 30℃; injection volume is 10μL; elution time is 55min; detector is ultraviolet detector.

[0176] The elution gradient process is:

[0177] 1) At elution time 0 min, the volume fraction of mobile phase A is 85%, and the volume fraction of mobile phase B is 15%;

[0178] 2) When the elution time is 45 min, the volume fraction of mobile phase A is 30%, and the volume fraction of mobile phase B is 70%;

[0179] 3) When the elution time is 46 min, the volume fraction of mobile phase A is 85%, and the volume fraction of mobile phase B is 15%;

[0180] 4) When the elution time is 55 min, the volume fraction of mobile phase A is 85% and the volume fraction of mobile phase B is 15%.

[0181] Under this method, Vibergron and its diastereomer (impurity B) completely overlap. This method is a purity test method for Mirabegron API and is not suitable for purity test of Vibergron API. Figure 11~Figure 12 shown.

[0182] 2. Methodological Validation

[0183] (1) Specificity verification: The separation degree between each impurity peak and the main component peak meets the detection requirements, the separation degree between each known impurity and the adjacent impurity meets the detection requirements, the blank solution does not interfere with the detection of the main peak and each impurity, and the specificity is good.

[0184] (2) Verification of detection limit and quantification limit: Under the experimental conditions, the impurity reference stock solution was diluted step by step to prepare different concentrations of each impurity reference solution, and the signal-to-noise ratio (S / N) of each impurity was measured by injection, and then the detection limit and quantification limit of each impurity were determined (the detection limit was calculated as S / N ≥ 3, and the quantification limit was calculated as S / N ≥ 10). The specific experimental results are shown in Tables 1 and 2.

[0185] Table 1 Detection limit test results

[0186]

[0187] Table 2 Results of limit of quantitation test (n=5)

[0188]

[0189] (3) Linear relationship verification. The verification results are shown in Table 3.

[0190] Table 3 Linearity test results

[0191]

[0192] It can be seen from Table 3 that the impurities have a good linear relationship within the limit concentration range of 50% to 200%.

[0193] (4) Accuracy verification

[0194] Test solution: Take an appropriate amount of Vibegron, dissolve it in solvent and dilute it to make a solution containing about 2.0 mg per 1 mL, which serves as the recovery background solution;

[0195] Impurity reference substance stock solution: Accurately weigh appropriate amounts of impurity A reference substance, impurity B reference substance, impurity E reference substance, impurity F reference substance, VSD23003-Q1, VSD23003-M1, and VSD23003-M4 reference substances, dissolve and dilute with solvent to prepare stock solutions containing approximately 0.02 mg of the above reference substances per 1 mL.

[0196] Accuracy solution: Take an appropriate amount of vibergolone and an appropriate amount of impurity reference stock solution, dissolve them in solvent and quantitatively dilute them to make a solution containing approximately 2.0 mg of vibergolone and approximately 2 ug of other impurities per 1 mL, and prepare a solution containing 100% impurity concentration level.

[0197] Accurately measure 5 μL of each of the above accuracy solutions and inject them into the liquid chromatograph respectively, record the chromatogram, record the peak area (the peak area of ​​the impurities in the test sample needs to deduct the background peak area of ​​the impurities in the test sample solution), calculate the recovery rate, and the experimental results are shown in Tables 4 to 11 below.

[0198] Recovery rate calculation formula: .

[0199] Table 4 Summary of impurity A recovery rates

[0200]

[0201] Table 5 Summary of impurity B recovery rates

[0202]

[0203] Table 6 Summary of impurity E recovery rates

[0204]

[0205] Table 7 Summary of impurity F recovery rates

[0206]

[0207] Table 8 Summary of impurity recovery rates of VSD23003-Q1

[0208]

[0209] Table 9 Summary of recovery rates of impurities VSD23003-M1

[0210]

[0211] Table 10 Summary of recovery rates of impurities VSD23003-M4

[0212]

[0213] Table 11 Summary of recovery rates of Vibegron (VSD23003)

[0214]

[0215] From the results in Tables 4 to 11, it can be seen that the recoveries of each impurity and vibergron are all in the range of 80% to 120%; and the RSD values ​​of the recoveries are all less than 5%, indicating that the method has high accuracy.

[0216] (5) Durability verification

[0217] Fine-tune the chromatographic conditions:

[0218] ① Adjust the flow rate: adjust the original flow rate of 1.0 mL / min to 0.9 mL / min, 1.1 mL / min. Other chromatographic conditions were the same as those in the embodiment for detection.

[0219] Conclusion: According to the above chromatographic conditions, the flow rate can be changed in the range of 0.9mL / min-1.1mL / min to achieve the desired separation effect, which has little effect on the results.

[0220] ② Adjust the injection volume: adjust the original injection volume of 5 μL to 3 μL and 7 μL, and perform the detection under the same other chromatographic conditions as in the embodiment.

[0221] Conclusion: According to the above chromatographic conditions, the injection volume can be changed in the range of 3μL-7μL to achieve the desired separation effect, and it has little effect on the results.

[0222] ③ Adjust the column temperature: adjust the original column temperature of 40°C to 38°C and 42°C, and perform the test under the same other chromatographic conditions as in the example.

[0223] Conclusion: According to the above chromatographic conditions, the column temperature can be changed in the range of 38℃-42℃ to achieve the desired separation effect, which has little effect on the results.

[0224] ④ Adjusting the concentration of dipotassium hydrogen phosphate: The original concentration of dipotassium hydrogen phosphate was 10 mmol / L, which was adjusted to 8 mmol / L and 12 mmol / L. Other chromatographic conditions were the same as those in the embodiment for detection.

[0225] Conclusion: According to the above chromatographic conditions, the concentration of potassium dihydrogen phosphate can be changed in the range of 8mmol / L-12mmol / L to achieve the desired separation effect without affecting the results.

[0226] ⑤ Adjust the wavelength: adjust the original wavelength of 250 nm to 248 nm and 252 nm, and perform detection under other chromatographic conditions the same as those in the embodiment.

[0227] Conclusion: According to the above chromatographic conditions, the wavelength can be changed in the range of 248nm-252nm to achieve the desired separation effect without affecting the results.

[0228] In summary, this method has strong specificity, high sensitivity, high linear relationship fit, accuracy and durability, which can meet the detection requirements and can be used for the detection and content determination of related substances in vibergolone raw materials.

[0229] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are exhaustively listed. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0230] For those skilled in the art, several modifications and improvements may be made without departing from the concept of the present invention, all of which belong to the protection scope of the present invention. The protection scope of the present invention shall be based on the attached claims.

Claims

1. A quality control method for Vibergron, characterized in that: The quantitative analysis of substances in the Vibegron API by high performance liquid chromatography includes the following steps: S1. Solution preparation Preparation of blank solvent: methylene diphosphonic acid aqueous solution and methanol are mixed evenly to obtain blank solvent; Preparation of test solution: Accurately weigh the Vibegron API to be tested, dissolve it in blank solvent and dilute it to make a solution containing 2.0 mg of the test solution per 1 mL; Preparation of control solution: Accurately measure the test solution and dilute it with blank solvent to obtain a control solution with a concentration of 2 μg / mL; Preparation of reference solution: accurately weigh the vibergolone reference substance, dissolve it in blank solvent and dilute it to make a reference solution containing 2.0 mg of vibergolone reference substance per 1 mL; Preparation of impurity reference substance stock solutions: Accurately weigh impurity A reference substance, impurity B reference substance, impurity E reference substance, impurity F reference substance, VSD23003-Q1, VSD23003-M1, and VSD23003-M4 reference substances, respectively, dissolve and dilute with blank solvent to prepare 0.02 mg of each impurity reference substance stock solution per 1 mL, and then gradually dilute to the required concentration; Among them, VSD23003-Q1 is ; VSD23003-M1 is ; VSD23003-M4 is ; Impurity A is ; Impurity B is ; Impurity E is ; Impurity F is ; S2. Quantitative analysis of substances in the Vibergron API was performed simultaneously by high performance liquid chromatography, the detector of the high performance liquid chromatograph was an ultraviolet detector, and the chromatographic conditions were as follows: phosphate aqueous solution and methanol were used as mobile phase A, and acetonitrile was used as mobile phase B; gradient elution was used; the flow rate of the mobile phase was 0.8 mL / min to 1.2 mL / min; the chromatographic column was a C18 or C8 column; the column temperature was 30°C to 45°C; the injection volume was 4 μL to 6 μL; and the elution time was 45 min; In step S2, the phosphate in the phosphate aqueous solution is dipotassium hydrogen phosphate; In step S2, the gradient elution is performed according to the following linear gradient: 1) When the elution time is 0 min, the volume fraction of mobile phase A is 100%; 2) When the elution time is 3 min, the volume fraction of mobile phase A is 100%; 3) When the elution time is 5 min, the volume fraction of mobile phase A is 78%, and the volume fraction of mobile phase B is 22%; 4) When the elution time is 21 min, the volume fraction of mobile phase A is 65%, and the volume fraction of mobile phase B is 35%; 5) When the elution time is 27 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%; 6) When the elution time is 39 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%; 7) When the elution time is 39.1 min, the volume fraction of mobile phase A is 100%; 8) When the elution time is 45 minutes, the volume fraction of mobile phase A is 100%.

2. A Vibergron quality control method according to claim 1, characterized in that: In step S1, the volume ratio of the methylene diphosphonic acid aqueous solution to methanol in the blank solvent is 1:(0.99-1.01), and the concentration of the methylene diphosphonic acid aqueous solution is 9.9-10.1 mmol / L.

3. A Vibergron quality control method according to claim 1, characterized in that: In step S2, in the mobile phase A, the volume ratio of the phosphate aqueous solution to methanol is 95:5, and the concentration of phosphate in the phosphate aqueous solution is 5 mmol / L to 20 mmol / L.

4. A Vibergron quality control method according to claim 1, characterized in that: In step S2, the mobile phase A consists of a dipotassium hydrogen phosphate aqueous solution and methanol, the volume ratio of the dipotassium hydrogen phosphate aqueous solution to methanol is 95:5, and the concentration of the dipotassium hydrogen phosphate aqueous solution is 10 mmol / L.

5. A Vibergron quality control method according to claim 1, characterized in that: In step S2, the chromatographic column is a C18 chromatographic column of 4.6 mm×250 mm and 5 μm.

6. A Vibergron quality control method according to claim 1, characterized in that: In step S2, the flow rate of the mobile phase is 1.0 mL / min.

7. A Vibergron quality control method according to claim 1, characterized in that: In step S2, the injection volume is 5 μL.

8. A Vibergron quality control method according to claim 1, characterized in that: In step S2, the column temperature is 40°C.

Images