CN116183759A - Detection method and application of related substances of pezopanib hydrochloride intermediate PZP-M1 - Google Patents
Detection method and application of related substances of pezopanib hydrochloride intermediate PZP-M1 Download PDFInfo
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Abstract
The invention relates to the technical field of chemical drug analysis methods, in particular to a detection method and application of related substances of a pezopanib hydrochloride intermediate PZP-M1. The detection method comprises the following steps: detecting the sample solution by adopting high performance liquid chromatography; the detection conditions of the high performance liquid chromatography comprise: the chromatographic column is octadecylsilane chemically bonded silica chromatographic column; the detection wavelength is 213-217 nm; the aqueous solution of potassium dihydrogen phosphate with the pH value of between 3.0 and 3.2 and the mol/L of between 0.009 and 0.011 is used as a mobile phase A and methanol is used as a mobile phase B for gradient elution. The 8 related substances can be separated at one time and efficiently under the high-performance liquid chromatography detection condition, and the high specificity, the repeatability, the accuracy and the like of the related substances are ensured to be separated from the effective components at one time.
Description
Technical Field
The invention relates to the technical field of chemical drug analysis methods, in particular to a detection method and application of related substances of a pezopanib hydrochloride intermediate PZP-M1.
Background
The pezopanib hydrochloride bulk drug has the structure shown as follows:
if the pezopanib hydrochloride is taken as a raw material medicine and contains impurities, the impurities or the conversion of the impurities are not likely to be brought into the later production in the process of producing the preparation, so that the quality of the finished product of the preparation is affected. Therefore, the impurity control of the intermediate and the finished product in each step of the pezopanib hydrochloride is important.
By analysing the following synthetic route, the impurities that may be present are: and (3) the impurities are finally researched as known impurities according to synthetic process routes and impurity control strategies, wherein the impurities are PZP-SM1, PZP-SM2, PZP-M1-imA, PZP-M1-imB, PZP-M1-imC, PZP-M1-imF, PZP-M1-imG and PZP-M1-imJ.
Y.Li et al, (Journal of Pharmaceutical and Biomedical Analysis (2010) 493-507) disclose a method by which to detect that PZP-M1-imC exhibits a split peak and cannot be effectively separated and accurate quantitative calculations cannot be performed. The specific map is shown in figure 1.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for detecting related substances of a pezopanib hydrochloride intermediate PZP-M1, which can be used for efficiently measuring 8 related substances of the pezopanib hydrochloride intermediate PZP-M1 at one time, and has strong specificity and good durability.
The invention also aims to provide application of the detection method of the related substances of the pezopanib hydrochloride intermediate PZP-M1 in quality control of raw materials of the pezopanib hydrochloride intermediate PZP-M1.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the method for detecting related substances of the pezopanib hydrochloride intermediate PZP-M1 comprises the following steps:
detecting the sample solution by adopting high performance liquid chromatography;
the PZP-M1 is N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine;
the detection conditions of the high performance liquid chromatography comprise:
the chromatographic column is octadecylsilane chemically bonded silica chromatographic column;
the detection wavelength is 213-217 nm;
performing gradient elution by taking 0.009-0.011 mol/L potassium dihydrogen phosphate aqueous solution with the pH value of 3.0-3.2 as a mobile phase A and methanol as a mobile phase B;
the gradient elution procedure includes:
in a specific embodiment of the present invention, the gradient elution procedure comprises:
in a specific embodiment of the present invention, the related substances include at least one of I, II, III, IV, V, VI, VII, and VIII; the structural formula is as follows:
wherein the related substancesI has the formula C 9 H 11 N 3 Molecular weight 161.21; the molecular formula of the related substance II is C 4 H 2 Cl 2 N 2 Molecular weight 148.98; the molecular formula of the related substance III is C 13 H 12 ClN 5 Molecular weight 273.72; the molecular formula of the related substance IV is C 13 H 12 ClN 5 Molecular weight 273.72; the molecular formula of the related substance V is C 13 H 13 N 5 O, molecular weight 255.28; the molecular formula of the related substance VI is C 22 H 22 N 8 Molecular weight 398.47; the molecular formula of the related substance VII is C 15 H 17 N 5 O, molecular weight 283.34; the molecular formula of the related substance VIII is C 12 H 10 ClN 5 The molecular weight was 259.70.
In a specific embodiment of the invention, the mobile phase A is an aqueous solution of potassium dihydrogen phosphate with a pH of 3.1 and a mol/L of 0.01. Wherein the mobile phase A is obtained by adjusting the pH of an aqueous solution of potassium dihydrogen phosphate by adopting phosphoric acid.
In a specific embodiment of the present invention, the detection wavelength is 214 to 216nm, preferably 215nm.
In a specific embodiment of the invention, the flow rate of the gradient elution is 0.9-1.1 mL/min, preferably 1mL/min.
In a specific embodiment of the present invention, the column temperature of the chromatographic column is 28 to 32 ℃, preferably 29 to 31 ℃, more preferably 30 ℃.
In a specific embodiment of the invention, the chromatography column is an Agilent 5TC-C18 (2) (4.6 mm. Times.250 mm,5 μm) or a performance equivalent chromatography column.
In a specific embodiment of the invention, the method further comprises: and calculating the contents of related substances I, II, III, IV, V, VI, VII and/or VIII in the test sample solution by a main component self-comparison method with correction factors. Specifically, the sample solution and the control solution are respectively injected into a high performance liquid chromatograph, the corresponding chromatographic peak area is measured under the detection condition of the high performance liquid chromatograph, and the contents of related substances I, II, III, IV, V, VI, VII and/or VIII in the sample solution are calculated by a self-contrast method.
In a specific embodiment of the present invention, the calculation formula is as follows:
single impurity calculation formula:
the total impurity calculation formula:
f: an impurity correction factor is known;
A known impurities : peak areas of known impurities in the chromatogram of the sample solution;
A impurity(s) : peak area of single impurity in chromatogram of sample solution;
A total impurities : the sum of peak areas of various impurities in a chromatogram of the sample solution (the impurities with correction factors need to be multiplied by the correction factors);
A control : main peak area in control solution chromatogram.
In a specific embodiment of the present invention, the preparation of the test solution comprises: methanol is adopted to dissolve the test sample. The test article comprises a pezopanib hydrochloride intermediate N- (2-chloropyrimidine-4-yl) -2, 3-dimethyl-2H-indazol-6-amine. In the preparation of the control solution and the impurity control solution, the adopted solvents are methanol.
In practice, the concentration of N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine in the test solution may be 0.5-2 mg/mL, for example, 1mg/mL. In practice, the concentration of N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine is not limited thereto.
In a specific embodiment of the present invention, the detection conditions include:
the chromatographic column is Agilent 5TC-C18 (2), 4.6mm×250mm,5 μm;
the column temperature of the chromatographic column is 30 ℃;
the detection wavelength is 215nm;
carrying out gradient elution by taking 0.01mol/L potassium dihydrogen phosphate aqueous solution with the pH value of 3.1 as a mobile phase A and methanol as a mobile phase B;
the flow rate of the gradient elution is 1mL/min.
The invention also provides an application of the detection method of any one of the related substances of the pezopanib hydrochloride intermediate PZP-M1 in quality control of N- (2-chloropyrimidine-4-yl) -2, 3-dimethyl-2H-indazole-6-amine.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the synthesis process of the pezopanib hydrochloride intermediate PZP-M1, the related substances I, II, III, IV, V, VI, VII and VIII contained in the intermediate are controlled, and the 8 related substances can be separated at one time and efficiently under the detection condition of the high performance liquid chromatography, so that the intermediate has good specificity, repeatability, accuracy and the like on the basis of ensuring the efficient separation of each related substance and the effective component;
(2) The detection method of the invention can be used for quality control of the pezopanib hydrochloride intermediate PZP-M1.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chromatogram obtained by detecting a sample solution by a method in the prior art;
FIG. 2 is a chromatogram of a blank solvent provided in example 1 of the present invention;
FIG. 3 is a chromatogram of a system applicability solution provided in example 1 of the present invention;
FIG. 4 is a chromatogram of a PZP-SM1 localization solution provided in example 1 of the present invention;
FIG. 5 is a chromatogram of a PZP-SM2 localization solution provided in example 1 of the present invention;
FIG. 6 is a chromatogram of a PZP-M1-imA positioning solution provided in example 1 of the present invention;
FIG. 7 is a chromatogram of a PZP-M1-imB positioning solution provided in example 1 of the present invention;
FIG. 8 is a chromatogram of a PZP-M1-imC positioning solution provided in example 1 of the present invention;
FIG. 9 is a chromatogram of a PZP-M1-imF positioning solution provided in example 1 of the present invention;
FIG. 10 is a chromatogram of a PZP-M1-imG positioning solution provided in example 1 of the present invention;
FIG. 11 is a chromatogram of a PZP-M1-imJ positioning solution provided in example 1 of the present invention;
FIG. 12 is a chromatogram of a sample solution provided in example 1 of the present invention;
FIG. 13 is a chromatogram of a system applicability solution provided in comparative example 1 of the present invention;
FIG. 14 is a chromatogram of a system applicability solution provided in comparative example 2 of the present invention;
FIG. 15 is a chromatogram of a PZP-M1-imJ positioning solution provided in comparative example 2 of the present invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and detailed description, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative of the present invention only and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the specific implementation mode, the system applicability, detection limit and quantitative limit, repeatability, linearity and range, accuracy, solution stability and other items are verified, and the method is verified according to the related guidelines in the annex of the national formulary of the State of the people's republic of China, namely, the technical guidelines of the standardization process established by the quality standards of chemical medicaments, the technical guidelines of the research on the impurities of chemical medicaments and the technical guidelines of the research on the residual solvents of chemical medicaments.
The sample solutions in the following examples were prepared mainly from N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine, and related substances I, II, III, IV, V, VI, VII, and VIII; in actual detection, the pezopanib hydrochloride intermediate N- (2-chloropyrimidine-4-yl) -2, 3-dimethyl-2H-indazole-6-amine to be detected is dissolved and diluted to prepare a test solution.
Example 1
The example provides a method for detecting a pezopanib hydrochloride intermediate N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine related substance, comprising the following steps:
(1) Sample preparation
Solvent: methanol;
blank solvent: methanol;
test solution: taking about 10mg of a sample to be measured, precisely weighing, placing the sample into a 10mL measuring flask, adding a solvent to dissolve and dilute the sample to a scale, and shaking the sample uniformly. (at a concentration of about 1 mg/mL)
Control solution: precisely measuring 1mL of the sample solution, placing the sample solution into a 100mL measuring flask, diluting the sample solution to a scale with a solvent, and shaking the sample solution uniformly; then precisely measuring 1mL, placing in a 10mL measuring flask, diluting to a scale with a solvent, and shaking uniformly. (at a concentration of about 1. Mu.g/mL)
Stock solution of PZP-SM1 (related substance I): about 10mg of PZP-SM1 reference substance is taken, precisely weighed, placed in a 50mL measuring flask, dissolved by adding a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.2 mg/mL)
PZP-SM1 localization solution: 1mL of PZP-SM1 stock solution is precisely measured, placed in a 100mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 2. Mu.g/mL)
Stock solution of PZP-SM2 (related substance II): about 10mg of PZP-SM2 reference substance is taken, precisely weighed, placed in a 20mL measuring flask, dissolved by a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.5 mg/mL)
PZP-SM2 localization solution: 1mL of PZP-SM2 stock solution is precisely measured, placed in a 100mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 5. Mu.g/mL)
Stock solution of PZP-M1-imB (related substance IV): about 10mg of PZP-M1-imB reference substance is taken, precisely weighed, placed in a 50mL measuring flask, dissolved by a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.2 mg/mL)
PZP-M1-imB positioning solution: 1mL of PZP-M1-imB stock solution is precisely measured, placed in a 10mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 20. Mu.g/mL)
Stock solution of PZP-M1-imA (related substance III): about 10mg of PZP-M1-imA reference substance is taken, precisely weighed, placed in a 100mL measuring flask, dissolved by a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.1 mg/mL)
PZP-M1-imA positioning solution: 1mL of PZP-M1-imA stock solution is precisely measured, placed in a 100mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 1. Mu.g/mL)
Stock solution of PZP-M1-imC (related substance V): about 10mg of PZP-M1-imC reference substance is taken, precisely weighed, placed in a 100mL measuring flask, dissolved by a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.1 mg/mL)
PZP-M1-imC positioning solution: 1mL of PZP-M1-imC stock solution is precisely measured, placed in a 100mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 1. Mu.g/mL)
Stock solution of PZP-M1-imF (related substance VI): about 10mg of PZP-M1-imF reference substance is taken, precisely weighed, placed in a 100mL measuring flask, dissolved by a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.1 mg/mL)
PZP-M1-imF positioning solution: 1mL of PZP-M1-imF stock solution is precisely measured, placed in a 100mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 1. Mu.g/mL)
Stock solution of PZP-M1-imG (related substance VII): about 10mg of PZP-M1-imG reference substance is taken, precisely weighed, placed in a 100mL measuring flask, dissolved by a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.1 mg/mL)
PZP-M1-imG positioning solution: 1mL of PZP-M1-imG stock solution is precisely measured, placed in a 100mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 1. Mu.g/mL)
Stock solution of PZP-M1-imJ (related substance VIII): about 10mg of PZP-M1-imJ reference substance is taken, precisely weighed, placed in a 100mL measuring flask, dissolved by a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.1 mg/mL)
PZP-M1-imJ positioning solution: 1mL of PZP-M1-imJ stock solution is precisely measured, placed in a 100mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 1. Mu.g/mL)
Impurity mixed solution: 1mL of each of the stock solutions of PZP-SM1, PZP-SM2, PZP-M1-imA, PZP-M1-imC, PZP-M1-imF, PZP-M1-imG and PZP-M1-imJ is precisely measured, placed in a 10mL measuring flask, diluted to a scale with a solvent, and shaken uniformly. (the concentration of PZP-SM1 is about 20. Mu.g/mL, the concentration of PZP-SM2 is about 50. Mu.g/mL, the concentrations of PZP-M1-imA, PZP-M1-imC, PZP-M1-imF, PZP-M1-imG, PZP-M1-imJ are about 10. Mu.g/mL, respectively)
System applicability solution: taking about 10mg of a sample to be measured, precisely weighing, placing the sample into a 10mL measuring flask, adding a proper amount of solvent to dissolve the sample, precisely adding 1mL of PZP-M1-imB stock solution and 1mL of impurity mixed solution, diluting the solution to a scale with the solvent, and shaking the solution uniformly. (PZP-SM 1 concentration is about 2. Mu.g/mL, PZP-SM2 concentration is about 5. Mu.g/mL, PZP-M1-imB concentration is about 20. Mu.g/mL, PZP-M1-imA, PZP-M1-imC, PZP-M1-imF, PZP-M1-imG, PZP-M1-imJ concentration is about 1. Mu.g/mL, respectively)
(2) High performance liquid chromatography detection conditions
Chromatographic column: agilent 5TC-C18 (2), 4.6mm.times.250 mm,5 μm;
mobile phase a:0.01mol/L of potassium dihydrogen phosphate aqueous solution, and regulating the pH value to 3.1 by phosphoric acid;
mobile phase B: methanol;
gradient elution was performed as in table 1;
flow rate: 1.0mL/min; detection wavelength: 215nm; column temperature: 30 ℃; sample injection amount: 10 mu L.
TABLE 1 gradient elution program table (volume fraction)
(3) Detection step
Taking 10 mu L of each of blank solvent, system applicability solution, positioning solution of each related substance, test sample solution, control solution and the like, carrying out sample injection detection, and recording a chromatogram. Wherein fig. 2 to 12 are chromatograms corresponding to different solutions, respectively.
Examples 2 to 14
Examples 2 to 14 each provide a different method for detecting a substance related to N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine, which is an intermediate of pezopanib hydrochloride, and the conditions of a certain parameter of the detection method in example 1 are changed. The specific change parameter information is shown in table 2.
Table 2 parameter information modified in examples 2 to 14
The test results of examples 1 to 14 are shown in tables 3 to 6.
TABLE 3 System applicability test results-retention time (min)
TABLE 4 System applicability test results-theoretical plate number
Table 5 System suitability test results-tailing factor
TABLE 6 System applicability test results-degree of separation
From the above results, it can be seen that:
under all conditions, the blank solvent does not interfere with the determination of the main component and impurities;
in the system applicability solution, the separation degree between a main peak and known impurities and adjacent impurity peaks is more than 1.5, the number of theoretical plates of the main peak and each known impurity is not less than 5000, and the tailing factor is more than 2.0;
therefore, the detection method can stably and effectively detect related substances of the pezopanib hydrochloride intermediate PZP-M1.
Comparative example 1
Comparative example 1 provides a detection method comprising the steps of:
comparative example 1 the detection method of reference example 1 is different in that: mobile phase a is different.
In comparative example 1, mobile phase A was an aqueous solution of 0.01mol/L potassium dihydrogen phosphate and the pH was adjusted to 5.4 with phosphoric acid. The chromatogram of the resulting system applicability solution is shown in FIG. 13.
As can be seen from the graph, the retention time of PZP-SM2 was consistent with that of PZP-M1-imC, the chromatographic peaks overlapped, the peak pattern of PZP-M1-imB was poor, and the peaks of PZP-M1-imG and PZP-M1-imF did not appear.
Comparative example 2
Comparative example 2 the system applicability solution of example 1 of the present invention was tested with reference to the method for detecting related impurities of intermediate ii described in example 2 of the patent application publication No. CN 107991398A. The chromatograms of the obtained system applicability solution and the PZP-M1-imJ positioning solution are shown in FIG. 14 and FIG. 15, respectively.
As is clear from the figure, the method of comparative example 2, namely PZP-M1 and PZP-M1-imJ, cannot be effectively separated and cannot be quantitatively calculated.
Experimental example 1
Sensitivity test
(1) Sample preparation
Solvent: methanol;
blank solvent: methanol;
stock solutions of various related substances, control solutions, system applicability solutions: the corresponding solutions prepared under "example 1" were used.
pZP-SM1 control solution: accurately measuring 2.5mL of PZP-SM1 stock solution, placing into a 10mL measuring flask, diluting to scale with solvent, and shaking. (at a concentration of about 50. Mu.g/mL)
pZP-M1 control solution: taking about 10mg of PZP-M1 reference substance, precisely weighing, placing into a 10mL measuring flask, adding solvent to dissolve and dilute to scale, and shaking uniformly; precisely measuring 1mL, placing in a 10mL measuring flask, diluting to a scale with a solvent, and shaking uniformly. (at a concentration of about 100. Mu.g/mL)
Quantitatively limiting stock solution: precisely measuring 2mL of PZP-M1 reference solution, 1mL of PZP-SM1 reference solution, 0.5mL of PZP-SM2 stock solution, 0.8mL of PZP-M1-imB stock solution, 3mL of PZP-M1-imA stock solution, 2mL of PZP-M1-imC stock solution, 2mL of PZP-M1-imG stock solution, 1.5mL of PZP-M1-imF stock solution and 1.5mL of PZP-M1-imJ stock solution, placing in the same 100mL measuring flask, diluting to scale with solvent, and shaking. (about containing PZP-M1 2. Mu.g/mL, PZP-SM 1.5. Mu.g/mL, PZP-SM 2.5. Mu.g/mL, PZP-M1-imB 1.6.6. Mu.g/mL, PZP-M1-imA. Mu.g/mL, PZP-M1-imC. Mu.g/mL, PZP-M1-imG. Mu.g/mL, PZP-M1-imF1.5. Mu.g/mL, PZP-M1-imJ 1.5.5. Mu.g/mL)
Quantitative limiting solution: precisely measuring 1mL of quantitative limited stock solution, placing into a 10mL measuring flask, diluting to a scale with a solvent, and shaking uniformly. (S/N. Apprxeq.10)
Detection limit solution: precisely measuring 4mL of quantitative limiting solution, placing the solution into a 10mL measuring flask, diluting the solution to a scale with a solvent, and shaking the solution uniformly. (S/N. Apprxeq.3)
(2) High performance liquid chromatography detection conditions: as in example 1.
(3) Detection step
Taking 10 mu L of each of a blank solvent (1 needle), a system applicability solution (1 needle), a quantitative limiting solution (6 needles) and a detection limiting solution (2 needles), sequentially injecting samples for detection, and recording a chromatogram.
(4) Analysis results: the sensitivity test results are shown in tables 7 to 17.
Table 7 results of System applicability solution detection
TABLE 8 PZP-SM1 quantitative limit test results
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 3.98 | 4.14 | 4.25 | 3.99 | 4.26 | 4.17 | 4.13 | 3.00 |
| Retention time (min) | 3.941 | 3.943 | 3.938 | 3.935 | 3.935 | 3.931 | 3.937 | 0.12 |
Table 9 PZP-M1-imC quantitative limit test results
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 9.94 | 10.37 | 10.45 | 9.87 | 10.20 | 10.15 | 10.16 | 2.25 |
| Retention time (min) | 8.470 | 8.480 | 8.470 | 8.458 | 8.459 | 8.459 | 8.466 | 0.11 |
TABLE 10 PZP-SM2 quantitative limit test results
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 5.83 | 5.67 | 5.68 | 5.60 | 5.99 | 5.56 | 5.72 | 2.80 |
| Retention time (min) | 11.375 | 11.377 | 11.356 | 11.360 | 11.350 | 11.350 | 11.361 | 0.11 |
Table 11 PZP-M1-imG quantitative limit test results
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 8.64 | 9.16 | 8.79 | 9.24 | 9.47 | 8.70 | 9.00 | 3.75 |
| Retention time (min) | 20.909 | 20.898 | 20.891 | 20.893 | 20.886 | 20.893 | 20.895 | 0.04 |
Table 12 PZP-M1-imF quantitative limit test results
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 7.62 | 7.93 | 7.81 | 7.42 | 7.79 | 7.78 | 7.72 | 2.35 |
| Retention time (min) | 28.855 | 28.844 | 28.853 | 28.845 | 28.836 | 28.839 | 28.845 | 0.03 |
TABLE 13 PZP-M1 quantitative limit test results
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 10.28 | 10.76 | 11.12 | 9.87 | 11.23 | 10.17 | 10.57 | 5.20 |
| Retention time (min) | 30.462 | 30.449 | 30.465 | 30.464 | 30.455 | 30.470 | 30.461 | 0.02 |
TABLE 14 quantitative limit test results for PZP-M1-imJ
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 6.51 | 6.52 | 6.68 | 6.03 | 6.52 | 6.05 | 6.39 | 4.28 |
| Retention time (min) | 33.343 | 33.338 | 33.365 | 33.367 | 33.355 | 33.358 | 33.354 | 0.04 |
Table 15 PZP-M1-imB quantitative limit test results
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 7.35 | 7.52 | 7.54 | 6.95 | 7.48 | 7.30 | 7.36 | 2.97 |
| Retention time (min) | 36.558 | 36.540 | 36.589 | 36.571 | 36.565 | 36.573 | 36.566 | 0.04 |
Table 16 PZP-M1-imA quantitative limit test results
| Name of the |
1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 14.32 | 14.15 | 13.74 | 13.19 | 14.17 | 12.94 | 13.75 | 4.15 |
| Retention time (min) | 41.804 | 41.793 | 41.849 | 41.829 | 41.823 | 41.852 | 41.825 | 0.06 |
TABLE 17 sensitivity test results
The calculation formula is as follows:
quantitative limit/detection limit concentration (μg/mL) =sample size×impurity content/dilution factor;
quantitative limit/detection limit (ng) =concentration×sample size;
the quantitative limit accounts for the test sample ratio (%) = quantitative limit concentration/test sample concentration x 100%;
from the above sensitivity test results, it can be seen that:
the quantitative limiting solution is continuously tested for 6 times, the peak areas RSD of the PZP-SM1 and the impurities are not more than 10.0%, and the retention time RSD is not more than 2.0%; the quantification limits were all less than the reported limit (0.05% test concentration).
Experimental example 2
Linearity test
(1) Sample preparation
Solvent: methanol;
blank solvent: a solvent;
control solution, system applicability solution: the corresponding solutions prepared under "example 1" were used.
Quantitative limiting solution: the "quantitative limiting solution" prepared under the item "Experimental example 1" was used.
pZP-SM1 solution: about 10mg of PZP-SM1 reference substance is taken, precisely weighed, placed in a 10mL measuring flask, dissolved by adding solvent, diluted to scale and shaken well. (about 1 mg/mL) (solutions of PZP-M1, PZP-SM2, PZP-M1-imB, PZP-M1-imA, PZP-M1-imC, PZP-M1-imG, PZP-M1-imJ were formulated in the same manner)
pZP-M1-imF solution: about 10mg of PZP-M1-imF reference substance is taken, precisely weighed, placed in a 100mL measuring flask, dissolved by a solvent, diluted to a scale and shaken uniformly. (at a concentration of about 0.1 mg/mL)
Linear stock solution: precisely measuring 1mL of each of PZP-M1 solution, PZP-SM2 solution, PZP-M1-imB solution, PZP-M1-imA solution, PZP-M1-imC solution, PZP-M1-imG solution and PZP-M1-imJ solution, precisely measuring 10mL of PZP-M1-imF solution, placing in the same 50mL measuring flask, diluting to scale with solvent, and shaking uniformly. (PZP-M1, PZP-SM2, PZP-M1-imB, PZP-M1-imA, PZP-M1-imC, PZP-M1-imF, PZP-M1-imG, PZP-M1-imJ concentrations are about 20 μg/mL, respectively)
Linear solution 1: the solution was limited quantitatively.
Linear solution 2 (equivalent to 0.05% of the sample concentration): precisely measuring 0.5mL of linear stock solution, placing the linear stock solution into a 20mL measuring flask, diluting to a scale with a solvent, and shaking uniformly. (at a concentration of about 0.5. Mu.g/mL)
Linear solution 3 (equivalent to 0.1% of the sample concentration): 1mL of the linear stock solution is precisely measured, placed in a 20mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 1. Mu.g/mL)
Linear solution 4 (equivalent to 0.2% of the sample concentration): 1mL of the linear stock solution is precisely measured, placed in a 10mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 2. Mu.g/mL)
Linear solution 5 (equivalent to 0.5% of the sample concentration): 2.5mL of the linear stock solution is precisely measured, placed in a 10mL measuring flask, diluted to a scale with a solvent, and shaken well. (at a concentration of about 5. Mu.g/mL)
Linear solution 6 (corresponding to 1.0% of the sample concentration): precisely measuring 5mL of linear stock solution, placing the linear stock solution into a 10mL measuring flask, diluting the linear stock solution to a scale with a solvent, and shaking the linear stock solution uniformly. (at a concentration of about 10. Mu.g/mL)
Linear solution 7 (equivalent to 2.0% of the sample concentration): precisely measuring PZP-M1-imB solution and PZP-M1 solution each 0.4mL, placing into 20mL measuring flask, diluting to scale with solvent, and shaking. (at a concentration of about 20. Mu.g/mL)
Linear solution 8 (corresponding to 4.0% of the sample concentration): precisely measuring the PZP-M1-imB solution and the PZP-M1 solution respectively by 0.8mL, placing into a 20mL measuring flask, diluting to scale with solvent, and shaking. (at a concentration of about 40. Mu.g/mL)
(2) High performance liquid chromatography detection conditions: as in example 1.
(3) Detection step
Taking 10 mu L of each of a blank solvent (1 needle), a system applicability solution (1 needle) and linear solutions (1 needle) and 8 needles, sequentially injecting samples for detection, and recording a chromatogram.
(4) Analysis results: the results of the linear tests are shown in tables 18 to 28.
Table 18 system applicability solution test results
TABLE 19 PZP-SM1 Linear test results
Table 20 PZP-M1-imC Linear test results
TABLE 21 PZP-SM2 Linear test results
Table 22 PZP-M1-imG Linear test results
TABLE 23 linear test results of PZP-M1-imF
TABLE 24 PZP-M1 Linear test results-1
TABLE 25 PZP-M1 Linear test results-2
TABLE 26 PZP-M1-imJ Linear test results
Table 28 results of the PZP-M1-imA Linear test
The linear correlation coefficient r is more than or equal to 0.990; the y-axis intercept is less than 25% of the peak area of the 100% limit concentration, and the RSD of the response factors is not more than 10.0%. And (3) taking the concentration C (mug/mL) as an abscissa and the corresponding peak area as an ordinate, so as to obtain a linear regression equation. The concentrations of PZP-SM1, PZP-M1-imC, PZP-SM2, PZP-M1-imG, PZP-M1-imF, PZP-M1-imJ, PZP-M1-imB and PZP-M1-imA have good linear relation with peak areas, and meet verification requirements.
Experimental example 3
Accuracy test
(1) Sample preparation
Solvent: methanol;
blank solvent: a solvent;
system applicability solution: the corresponding solutions prepared under "example 1" were used.
pZP-SM1 control stock solution: about 10mg of PZP-SM1 reference substance is taken, put into a 10mL measuring flask, dissolved and diluted to the scale by a solvent, shaken uniformly and prepared in parallel to 2 parts. (at a concentration of about 1 mg/mL) (preparation of PZP-SM2, PZP-M1-imA, PZP-M1-imC, PZP-M1-imF, PZP-M1-imG, PZP-M1-imJ control stock solutions) in the same manner
pZP-M1-imB control stock solution: about 20mg of PZP-M1-imB reference substance is taken, put into a 20mL measuring flask, dissolved and diluted to a scale by a solvent, and shaken uniformly. (at a concentration of about 1 mg/mL)
pZP-M1-imF control stock solution: about 10mg of PZP-M1-imF reference substance is taken, put into a 100mL measuring flask, dissolved and diluted to a scale by a solvent, and shaken uniformly. (at a concentration of about 0.1 mg/mL)
Mixing the control stock solution: precisely measuring 1mL of the PZP-SM1 control stock solution, 2.5mL of the PZP-SM2 control stock solution, 10mL of the PZP-M1-imB control stock solution, 0.5mL of each of the PZP-M1-imC, the PZP-M1-imG and the PZP-M1-imJ control stock solution, precisely measuring 5mL of the PZP-M1-imF control stock solution, placing the same 50mL measuring flask, diluting to a scale with a solvent, and shaking uniformly. (PZP-SM 1. Mu.g/mL, PZP-SM2 50. Mu.g/mL, PZP-M1-imB. Mu.g/mL, PZP-M1-imA, PZP-M1-imC, PZP-M1-imF, PZP-M1-imG and PZP-M1-imJ each have a concentration of about 10. Mu.g/mL, and two portions are prepared in parallel in one-to-one correspondence with a reference stock solution)
Control solution: precisely measuring 1mL of mixed reference stock solution, placing into a 10mL measuring flask, diluting to scale with solvent, and shaking. (PZP-SM 1 2. Mu.g/mL, PZP-SM2 5. Mu.g/mL, PZP-M1-imB. Mu.g/mL, PZP-M1-imA, PZP-M1-imC, PZP-M1-imF, PZP-M1-imG and PZP-M1-imJ each have a concentration of about 1. Mu.g/mL, and two portions are prepared in parallel in one-to-one correspondence with a reference stock solution)
Test solution: about 10mg of the product is taken, precisely weighed, placed in a 10mL measuring flask, dissolved by adding a solvent, diluted to a scale and shaken well. (at a concentration of about 1 mg/mL)
Recovery solution (mixing reference stock solution-1 preparing recovery solution)
50% recovery solution: about 10mg of the product is taken, precisely weighed, placed in a 10mL measuring flask, added with a proper amount of solvent to dissolve, precisely added with 0.5mL of reference stock solution, diluted to a scale with the solvent, and shaken well. (parallel preparation of 3 parts)
100% recovery solution: about 10mg of the product is taken, precisely weighed, placed in a 10mL measuring flask, added with a proper amount of solvent to dissolve, precisely added with 1mL of reference substance stock solution, diluted to a scale with the solvent, and shaken well. (parallel preparation of 3 parts)
150% recovery solution: about 10mg of the product is taken, precisely weighed, placed in a 10mL measuring flask, added with a proper amount of solvent to dissolve, precisely added with 1.5mL of reference stock solution, diluted to a scale with the solvent, and shaken well. (parallel preparation of 3 parts)
(2) High performance liquid chromatography detection conditions: as in example 1.
(3) Detection step
Taking 10 mu L of each of a blank solvent (1 needle), a system applicability solution (1 needle), a reference substance solution-1 (continuous 5 needles), a reference substance solution-2 (2 needles), a test substance solution (1 needle), a 50% solution-1 (1 needle), a 50% solution-2 (1 needle), a 50% solution-3 (1 needle), a 100% solution-1 (1 needle), a 100% solution-2 (1 needle), a 100% solution-3 (1 needle), a 150% solution-1 (1 needle), a 150% solution-2 (1 needle) and a 150% solution-3 (1 needle), sequentially sampling, detecting and recording a chromatogram.
(4) Analysis results
The calculation formula is as follows: percent recovery = (measured-content in sample)/addition x 100%. The test results are shown in tables 29 to 37.
Table 29 system applicability solution test results
TABLE 30 PZP-SM1 recovery test results
TABLE 31 recovery test results of PZP-M1-imC
TABLE 32 PZP-SM2 recovery test results
TABLE 33 recovery test results of PZP-M1-imG
TABLE 34 recovery test results of PZP-M1-imF
Table 35 recovery test results of PZP-M1-imJ
TABLE 36 recovery test results of PZP-M1-imB
TABLE 37 recovery test results of PZP-M1-imA
The impurity limit concentration is taken as 100%, the impurities are quantitatively added into the sample according to three concentrations of 50%, 100% and 150%, the impurity recovery rate is in the range of 90.0% -110.0% at each concentration level, and the 9-part recovery rate RSD is less than 5.0%, so that the accuracy is good.
Experimental example 4
Repeatability test
(1) Sample preparation
Solvent: methanol;
blank solvent: a solvent;
system applicability solution: the corresponding solutions prepared under "example 1" were used.
Test solution: about 10mg of the product is taken, precisely weighed, placed in a 10mL measuring flask, dissolved by adding a solvent, diluted to a scale, and shaken uniformly to serve as a sample solution. (parallel preparation of 6 parts)
Control solution: precisely measuring 1mL of the sample solution, placing the sample solution into a 100mL measuring flask, diluting the sample solution to a scale with a solvent, shaking the sample solution uniformly, precisely measuring 1mL of the sample solution, placing the sample solution into a 10mL measuring flask, diluting the sample solution to the scale with the solvent, shaking the sample solution uniformly, and taking the sample solution as a control solution. (parallel preparation of 6 parts)
(2) High performance liquid chromatography detection conditions: as in example 1.
(3) Detection step
Taking 10 mu L of blank solvent (1 needle), system applicability solution (1 needle), test sample solutions 1-6 (1 needle respectively) and control solutions 1-6 (1 needle respectively), sequentially injecting samples for detection, and recording a chromatogram.
(4) Analysis results
The results of the repeatability test are shown in tables 38 to 39 below.
Table 38 system applicability solution test results
Table 39 repeatability test results
6 parts of test samples, basically consistent impurity detection, no obvious change of total impurities and impurity number and good repeatability.
Experimental example 5
Solution stability test
(1) Sample preparation
Solvent: methanol;
blank solvent: a solvent;
system applicability solution: the corresponding solutions prepared under "example 1" were used;
the test solution and the control solution were prepared as in example 1.
(2) High performance liquid chromatography detection conditions: as in example 1.
(3) Detection step
Taking 10 mu L of each of blank solvent, system applicability solution, control solution, test sample solution and the like, carrying out sample injection detection according to a table 40, and recording a chromatogram.
Table 40 sample injection order and requirements
| Sequence number | Sample of | Needle count | Sequence number | Sample of | Needle count |
| 1 | Blank solvent | 1 needle | 19 | Control solution 1 | 1 needle |
| 2 | System applicability solution | 1 needle | 20 | Test solution for 2-16h | 1 needle |
| 3 | Control solution 1 | Continuous 5-needle | 21 | Control solution 2-17h | 1 needle |
| 4 | Test solution for 2-0h | 1 needle | 22 | Blank solvent | 1 needle |
| 5 | Control solution 2-1h | 1 needle | 23 | Control solution 1 | 1 needle |
| 6 | Test solution for 2-2h | 1 needle | 24 | Test solution for 2-20h | 1 needle |
| 7 | Control solution 2-3h | 1 needle | 25 | Control solution 2-21h | 1 needle |
| 8 | Test solution for 2-4h | 1 needle | 26 | Blank solvent | 1 needle |
| 9 | Control solution for 2-5h | 1 needle | 27 | Control solution 1 | 1 needle |
| 10 | Control solution 1 | 1 needle | 28 | Test solution for 2-24h | 1 needle |
| 11 | Blank solvent | 1 needle | 29 | Control solution for 2-25h | 1 needle |
| 12 | Test solution for 2-8h | 1 needle | 30 | Blank solvent | 1 needle |
| 13 | Control solution 2-9h | 1 needle | 31 | Control solution 1 | 1 needle |
| 14 | Blank solvent | 1 needle | 32 | Test solution for 2-28h | 1 needle |
| 15 | Control solution 1 | 1 needle | 33 | Control solution 2-29h | 1 needle |
| 16 | Test solution for 2-12h | 1 needle | 34 | Blank solvent | 1 needle |
| 17 | Control solution 2-13h | 1 needle | 35 | Control solution 1 | 1 needle |
| 18 | Blank solvent | 1 needle |
(4) Analysis results: the test results are shown in tables 41 to 43.
Table 41 system applicability solution test results
TABLE 42 stability results of test solutions
Table 43 control solution stability results
| Time (h) | Retention time (min) | |
| 1 | 30.299 | 53.05 |
| 3 | 30.388 | 52.97 |
| 5 | 30.323 | 52.05 |
| 9 | 30.503 | 52.46 |
| 13 | 30.422 | 52.44 |
| 17 | 30.419 | 53.98 |
| 21 | 30.413 | 52.33 |
| 25 | 30.413 | 53.88 |
| 29 | 30.400 | 53.35 |
| Mean value of | 30.398 | 52.94 |
| RSD(％) | 0.20 | 1.30 |
The sample solution is placed for 28 hours at room temperature, and impurities PZP-M1-imC, PZP-M1-imG and PZP-M1-imA are not detected; the average content value of the impurity PZP-SM1 at each time point is 0.04%, and the impurity PZP-SM1 is relatively consistent; the average content value of the impurity PZP-SM2 at each time point is 0.10%, and RSD is 2.51% and less than 15.0%; the average content value of the impurity PZP-M1-imF at each time point is 0.04%, and the impurity PZP-M1-imF is relatively consistent; the average content of impurities PZP-M1-imJ at each time point is 0.05%, and RSD is 2.76% and less than 20.0%; the average content of the impurity PZP-M1-imB at each time point is 0.70%, the RSD is 0.85% and less than 5.0%; the average content of the total impurities at each time point is 1.28%, the RSD is 1.08% and less than 5.0%; the number of impurities is not changed obviously, which indicates that the solution of the test sample is stable after being placed for 28 hours at room temperature. The control solution was left at room temperature for 29h with a main peak area RSD of 1.30% or less than 5.0%, indicating that the control solution was stable at room temperature for 29 h.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. The method for detecting the related substances of the pezopanib hydrochloride intermediate PZP-M1 is characterized by comprising the following steps:
detecting the sample solution by adopting high performance liquid chromatography;
the PZP-M1 is N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine;
the detection conditions of the high performance liquid chromatography comprise:
the chromatographic column is octadecylsilane chemically bonded silica chromatographic column;
the detection wavelength is 213-217 nm;
performing gradient elution by taking 0.009-0.011 mol/L potassium dihydrogen phosphate aqueous solution with the pH value of 3.0-3.2 as a mobile phase A and methanol as a mobile phase B;
the gradient elution procedure includes:
2. the method for detecting a substance related to a pezopanib hydrochloride intermediate PZP-M1 according to claim 1, wherein the gradient elution procedure comprises:
3. the method for detecting a related substance of a pezopanib hydrochloride intermediate PZP-M1 according to claim 1, wherein the related substance comprises at least one of i, ii, iii, iv, v, vi, vii and viii; the structural formula is as follows:
4. the method for detecting a related substance of a pezopanib hydrochloride intermediate PZP-M1 according to claim 1, wherein the mobile phase a is an aqueous solution of 0.01mol/L potassium dihydrogen phosphate having a pH of 3.1.
5. The method for detecting related substances of a pezopanib hydrochloride intermediate PZP-M1 according to claim 1, wherein the flow rate of the gradient elution is 0.9-1.1 mL/min;
preferably, the flow rate of the gradient elution is 1mL/min.
6. The method for detecting related substances of a pezopanib hydrochloride intermediate PZP-M1 according to claim 1, wherein the column temperature of the chromatographic column is 28-32 ℃;
preferably, the column temperature is 29-31 ℃;
more preferably, the column temperature is 30 ℃.
7. The method for detecting a substance related to a pezopanib hydrochloride intermediate PZP-M1 according to claim 1, wherein the method further comprises: and calculating the contents of related substances I, II, III, IV, V, VI, VII and/or VIII in the test sample solution by a main component self-comparison method with correction factors.
8. The method for detecting a substance related to a pezopanib hydrochloride intermediate PZP-M1 according to claim 1, wherein the preparation of the test solution comprises: dissolving a test sample by adopting methanol;
preferably, the test article comprises the intermediate N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine of pezopanib hydrochloride.
9. The method for detecting a substance related to a pezopanib hydrochloride intermediate PZP-M1 according to claim 1 or 2, wherein the detection conditions comprise:
the chromatographic column is Agilent 5TC-C18 (2), 4.6mm×250mm,5 μm;
the column temperature of the chromatographic column is 30 ℃;
the detection wavelength is 215nm;
carrying out gradient elution by taking 0.01mol/L potassium dihydrogen phosphate aqueous solution with the pH value of 3.1 as a mobile phase A and methanol as a mobile phase B;
the flow rate of the gradient elution is 1mL/min.
10. Use of the assay according to any one of claims 1 to 9 for quality control of N- (2-chloropyrimidin-4-yl) -2, 3-dimethyl-2H-indazol-6-amine.
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