CN115112777A - Regorafenib intermediate related substance analysis method - Google Patents
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Abstract
The invention provides a regorafenib intermediate related substance analysis method, which adopts reversed-phase high performance liquid chromatography to separate and measure regorafenib intermediate I and formula II compounds, formula III compounds, formula IV compounds, formula V compounds, formula VI compounds, formula VII compounds, formula VIII compounds, formula IX compounds, formula X compounds, formula XI compounds and formula XII compounds. The method has the advantages that the solvent does not interfere with impurity detection, the method is good in specificity, simple and convenient, high in sensitivity, good in repeatability and good in accuracy, qualitative and quantitative analysis of related substances of a test sample can be rapidly and accurately carried out, and the controllability of the quality of the regorafenib intermediate I is guaranteed.
Description
Technical Field
The invention relates to the field of drug analysis, in particular to a regorafenib intermediate related substance analysis method.
Background
Regorafenib is a novel oral multi-kinase inhibitor for the treatment of patients with metastatic colorectal cancer (CRC), gastrointestinal stromal tumor (GIST), and hepatocellular carcinoma (HCC). The medicine has been approved for use in 90 countries and regions such as the United states, European Union, Japan, etc., and is approved for marketing by CFDA 24.3.2017, and can be used for treating patients with metastatic colorectal cancer and gastrointestinal stromal tumor.
The chemical name of regorafenib is 4- [4- ({ [ 4-chloro-3- (trifluoromethyl) phenyl)]Carbamoyl } amino) -3-fluorophenoxy]-N-methylpyridine-2-carboxamide monohydrate of formula C 21 H 15 ClF 4 N 4 O 3 ·H 2 O, molecular weight is 500.83, and regorafenib is targeted to tumor generation, tumor angiogenesis and maintenance of tumor microenvironment signal transduction by inhibiting various protein kinases for promoting tumor growth. For the treatment of metastatic colorectal cancer; patients with locally advanced, inoperable resections or metastatic gastrointestinal stromal tumor (GIST) who had previously received treatment with imatinib and sunitinib.
In the preparation of regorafenib intermediates
When, the following impurities may be involvedWhich may have an impact on product quality, there is therefore a great need for an analytical method which allows the residual amount of the compounds of the formulae II to XII in the compound of formula I to be monitored and thus the risk of their transfer into regorafenib in the form of prototypes or structurally similar conductive impurities.
Disclosure of Invention
The invention provides a regorafenib intermediate related substance analysis method, wherein the result of the regorafenib intermediate is shown as a formula I.
In order to achieve the purpose, the technical scheme of the invention is as follows: a regorafenib intermediate related substance analysis method is characterized by comprising the following steps: the method is a high performance liquid chromatography, and the method adopts a reversed phase chromatographic column, takes a mixed solution of potassium dihydrogen phosphate buffer solution and acetonitrile as an eluent, and elutes according to isocratic or gradient.
In some embodiments, the present invention provides a regorafenib intermediate related substance assay method, characterized in that: the method is high performance liquid chromatography, and the method adopts a reversed phase chromatographic column and takes a mobile phase A and/or a mobile phase B as eluent, wherein the mobile phase A is potassium dihydrogen phosphate buffer solution-acetonitrile, and the mobile phase B is acetonitrile.
In the mobile phase A, the volume ratio of the potassium dihydrogen phosphate buffer solution to the acetonitrile is 95: 5.
in some more typical embodiments, the gradient elution is performed with potassium dihydrogen phosphate buffer-acetonitrile (95:5) as mobile phase a and acetonitrile as mobile phase B according to the following table:
in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of the mobile phase A is the percentage of the volume of the mobile phase A to the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the volume of the mobile phase B to the total volume of the eluent.
In some embodiments, the concentration of the potassium dihydrogen phosphate buffer is 0.005mol/L to 0.2 mol/L; in some typical embodiments, the concentration of the potassium dihydrogen phosphate buffer is 0.02mol/L to 0.04 mol/L; in some more typical embodiments, the concentration of the potassium dihydrogen phosphate buffer is 0.02 mol/L.
In some embodiments, the flow rate of the eluent is 0.5-2 mL/min; in some exemplary embodiments, the eluent has a flow rate selected from the group consisting of 0.5 to 1 mL/min; in some more typical embodiments, the flow rate of the eluent is 0.9 mL/min.
In some embodiments, the reverse phase chromatography column employs a non-polar stationary phase as packing; in some typical embodiments, the reverse phase chromatography column employs phenylsilane-bonded silica as packing; in some more typical embodiments, the reverse phase chromatography column is Kromasil100-5Phenyl, which is 250mm by 4.6mm, 5 μm in size.
In some embodiments, the analytical method is performed on a high performance liquid chromatograph, employing a diode array detector, an ultraviolet detector, a differential refraction detector, an electrospray detector, or an evaporative light scattering detector; in some exemplary embodiments, the analytical method is performed on a high performance liquid chromatograph, using a diode array detector or an ultraviolet detector; in some more typical embodiments, the analysis method is performed on a high performance liquid chromatograph, an ultraviolet detector is used, and the detection wavelength is 195nm to 400 nm; preferably 200nm to 322 nm; more preferably 230 nm.
In some embodiments, the reverse phase chromatography column has a column temperature of 20 to 55 ℃; in some typical embodiments, the column temperature of the reverse phase chromatography column is 20 to 30 ℃; in some more typical embodiments, the column temperature of the reverse phase chromatography column is 25 ℃.
On the other hand, the invention provides an analysis method of related substances of a regorafenib intermediate, which is characterized by comprising the following steps:
the analysis method is carried out on a high performance liquid chromatograph; the method adopts a reverse phase chromatographic column, and the reverse phase chromatographic column adopts phenyl silane bonded silica gel as a filler;
the analysis method adopts an ultraviolet detector, and the detection wavelength is 230 nm;
the analysis method adopts a reversed phase chromatographic column, and the column temperature is 25 ℃;
the analysis method uses a mobile phase A and/or a mobile phase B as an eluent, wherein the mobile phase A is potassium dihydrogen phosphate buffer solution-acetonitrile, the mobile phase B is acetonitrile, the potassium dihydrogen phosphate buffer solution is 0.02mol/L potassium dihydrogen phosphate aqueous solution, and the volume ratio of the potassium dihydrogen phosphate aqueous solution to the acetonitrile in the mobile phase A is 95: 5;
gradient elution was performed according to the following procedure:
in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of the mobile phase A is the percentage of the volume of the mobile phase A in the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the volume of the mobile phase B in the total volume of the eluent;
the flow rate of the eluent is 0.9 mL/min;
respectively injecting a test solution, a reference solution and a system applicability solution of the compound of the formula I;
the control solution comprises a compound of formula I and one or more of a compound of formula II, a compound of formula III, a compound of formula IV, a compound of formula V, a compound of formula VI, a compound of formula VII, a compound of formula VIII, a compound of formula IX, a compound of formula X, a compound of formula XI, and a compound of formula XII;
the system suitability solution comprises one or more mixtures of a compound of formula I and a compound of formula II, a compound of formula III, a compound of formula IV, a compound of formula V, a compound of formula VI, a compound of formula VII, a compound of formula VIII, a compound of formula IX, a compound of formula X, a compound of formula XI, a compound of formula XII;
the content of the compounds of the formula II-XII in the test sample is calculated by an external standard method.
In some specific embodiments, the present invention provides a method for analyzing a related substance of a regorafenib intermediate, comprising the following steps:
optionally, comprising the step of (1) formulating a positioning solution of a compound of formula II: taking a compound reference substance of the formula II, precisely weighing, adding acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), dissolving and diluting, fixing the volume, shaking up to obtain the compound,
wherein, the structural formula of the compound of formula II is:
optionally, comprising the step (2) of formulating a positioning solution of a compound of formula III: taking a compound of formula III as a reference substance, precisely weighing, adding acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), dissolving and diluting, fixing the volume, shaking up to obtain the compound,
wherein, the structural formula of the compound of the formula III is as follows:
optionally, comprising step (3) formulation of a compound positioning solution of formula IV: precisely weighing compound of formula IV, dissolving in acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), diluting to desired volume, shaking,
wherein, the structural formula of the compound of formula IV is as follows:
optionally, comprising step (4) formulation of a compound positioning solution of formula V: precisely weighing compound of formula V, dissolving in acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), diluting to desired volume, shaking,
wherein, the structural formula of the compound of the formula V is as follows:
optionally, comprising step (5) formulating a positioning solution of a compound of formula VI: precisely weighing compound of formula VI, dissolving in acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), diluting to desired volume, shaking,
wherein, the structural formula of the compound of formula VI is as follows:
optionally, comprising step (6) formulation of a localization solution of a compound of formula VII: precisely weighing compound of formula VII, dissolving in acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), diluting to desired volume, shaking,
wherein, the compound of formula VII has a structural formula:
optionally, comprising the step (7) of formulating a positioning solution of a compound of formula VIII: precisely weighing compound of formula VIII, dissolving in acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), diluting to desired volume, shaking,
wherein, the compound of formula VIII has the structural formula:
optionally, comprising the step (8) of formulating a positioning solution of a compound of formula IX: taking a compound of formula IX, precisely weighing, adding acetonitrile-water-trifluoroacetic acid (V: V: V is 200: 800: 0.5), dissolving and diluting, fixing the volume, shaking up to obtain the final product,
wherein said formula IXA compound having the structural formula:
optionally, comprising the step (9) of formulating a positioning solution for the compound of formula X: taking a compound of the formula X as a reference substance, precisely weighing, adding acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), dissolving and diluting, fixing the volume, shaking up uniformly to obtain the compound,
wherein, the compound of the formula X has a structural formula:
optionally, comprising step (10) of formulating a compound of formula XI: taking a compound of formula XI as a reference substance, precisely weighing, adding acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), dissolving and diluting, fixing the volume, shaking up to obtain the compound,
wherein, the compound of formula XI has a structural formula:
optionally, comprising step (11) of formulating a defined solution of a compound of formula XII: taking a compound reference substance of the formula XII, precisely weighing, adding acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), dissolving and diluting, fixing the volume, shaking up to obtain the compound,
wherein, the structural formula of the compound of the formula XII is:
optionally, comprising the step (12) of formulating a compound-positioning solution of formula I: taking a compound reference substance of the formula I, precisely weighing, adding acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5), dissolving and diluting, fixing the volume, shaking up to obtain the compound,
wherein, the structural formula of the compound of formula I is:
(13) preparing a system applicability solution: precisely weighing appropriate amount of compound of formula I and related substance compound, mixing, adding mixed solution of acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5) as solvent, dissolving and diluting, metering volume, and shaking; the related substance compound is selected from one or more of a compound of formula II, a compound of formula III, a compound of formula IV, a compound of formula V, a compound of formula VI, a compound of formula VII, a compound of formula VIII, a compound of formula IX, a compound of formula X, a compound of formula XI and a compound of formula XII;
(14) preparing a test solution: taking a proper amount of a sample, precisely weighing, taking a mixed solution of acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5) as a solvent for dissolving and diluting, fixing the volume, and shaking up uniformly to obtain the product;
(15) preparing a reference substance mixed stock solution: respectively taking appropriate amount of compound of formula I and related substance compound reference, precisely weighing, adding mixed solution of acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5) as solvent, dissolving and diluting, metering volume, and shaking; the related substance compound is selected from one or more of a compound of formula II, a compound of formula III, a compound of formula IV, a compound of formula V, a compound of formula VI, a compound of formula VII, a compound of formula VIII, a compound of formula IX, a compound of formula X, a compound of formula XI and a compound of formula XII;
(16) preparing a reference substance solution: precisely measuring a proper amount of the reference substance mixed stock solution, and quantitatively diluting with a mixed solution of acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5) as a solvent;
(17) and (3) testing the test sample: using Kromasil100-5Phenyl (4.6 mm. times.250 mm, 5 μm) chromatographic column; using 20mmol/L potassium dihydrogen phosphate buffer solution-acetonitrile (95:5) as mobile phase A, acetonitrile as mobile phase B, and adjusting the flow rate of eluent to 0.9ml per minute; the column temperature was 25 ℃; an ultraviolet detector is adopted as a detector, and the detection wavelength is 230 nm; the sample injection volume is 5 mul; respectively measuring mobile phase solution, positioning solution, system applicability solution, test solution and reference solution, respectively injecting into a liquid chromatograph, and performing gradient elution according to the following table;
in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of the mobile phase A is the percentage of the volume of the mobile phase A in the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the volume of the mobile phase B in the total volume of the eluent; recording each spectrogram;
(18) and (3) calculating the content:
wherein: the correction factor f ═ Cs/As;
cs is the concentration of each impurity of the reference solution, mu g/mL;
as-peak area of each impurity of the control solution;
at is the peak area of each impurity in the test solution;
n is the volume of the dilution multiple of the solvent of the test solution, mL;
wt is the sample weighing and mg of the sample;
the method includes at least any one of the steps (1) to (11).
Those skilled in the art can easily understand that, according to different purposes, the compounds for preparing the positioning solution in the steps (1) to (11), and the related substances in the steps (13) and (15) have corresponding relations; specifically, for example, when the amount of the compound of formula II in the test sample is determined, the method at least comprises the step (1), and the substances involved in the steps (13) and (15) at least comprise the compound of formula II; for another example, when the amounts of the compound of formula III and the compound of formula IV in the test sample are simultaneously determined, the method at least comprises the steps (2) and (3), and the related substances in the steps (13) and (15) at least comprise the compound of formula III and the compound of formula IV.
In some embodiments, the assay comprises both steps (1) through (11), and the compounds of interest in steps (13) and (15) are one or more of compounds of formula II, compounds of formula III, compounds of formula IV, compounds of formula V, compounds of formula VI, compounds of formula VII, compounds of formula VIII, compounds of formula IX, compounds of formula X, compounds of formula XI, compounds of formula XII.
It is easily understood by those skilled in the art that in the process of carrying out the present invention, those skilled in the art can appropriately adjust the sequence of the above steps according to actual requirements without affecting the implementation of the determination method, such as the configuration sequence of the positioning solution, the control solution, the system applicability solution and the test solution.
In some embodiments, the test solution contains no more than 0.5%, preferably no more than 0.2% of the compound of formula II; the content of the compound of the formula III is not higher than 0.5%, preferably not higher than 0.2%; the compound of formula IV is not higher than 0.5%, preferably not higher than 0.2%; the compound of formula V is not higher than 0.5%, preferably not higher than 0.2%; the compound of formula VI is not more than 0.5%, preferably not more than 0.2%; the compound of formula VII is not higher than 0.5%, preferably not higher than 0.2%; the compound of formula VIII is not higher than 0.5%, preferably not higher than 0.2%; no more than 0.5%, preferably no more than 0.2% of the compound of formula IX; the compound of formula X is not higher than 1.5%, preferably not higher than 1.0%; the compound of formula XI is not higher than 3.0%, preferably not higher than 1.5%; the compound of formula XII is not more than 0.5%, preferably not more than 0.2%.
In some embodiments, the positioning solution concentration is between 0.5 μ g/mL and 50 μ g/mL; preferably 2.5 to 10 mug/mL; more preferably 5. mu.g/mL.
In some embodiments, the concentrations of the compounds of formulae II-XII in the system-suitable solution are each independently: 0.5 mu g/mL-50 mu g/mL; preferably 2.5 to 10 mug/mL; more preferably 5. mu.g/mL.
In some embodiments, the concentration of the compound of formula I in the system suitability solution is: 0.1 mg/mL-2.5 mg/mL; preferably 0.25mg/mL to 1 mg/mL; more preferably 0.5 mg/mL.
In some embodiments, the concentration of the compound of formulae I-XII in the control solution is each independently: 0.5 mu g/mL-50 mu g/mL; preferably 2.5 to 10 mug/mL; more preferably 5. mu.g/mL.
In some embodiments, the present invention provides the use of a compound of formula V for the preparation of an impurity control for regorafenib intermediate related substance assays.
In some embodiments, the present invention provides a method of preparing a compound of formula V, comprising the steps of:
under the acidic condition, the compound of the formula II and the compound of the formula III are refluxed by ethanol to prepare the compound of the formula V.
In some embodiments, the present invention provides the use of a compound of formula XII for the preparation of an impurity control for regorafenib intermediate related substance assays.
In some embodiments, the present invention provides a process for preparing a compound of formula XII, comprising the steps of:
the compound in the formula II and the compound in the formula XI are prepared into the compound in the formula XII under the action of potassium tert-butoxide.
As used herein, the compound of formula II has the formula
The compounds may be obtained by direct purchase or by reference to published literature including, but not limited to, WO9932463a 1.
Here, the compound of formula III has the formula
The compounds may be obtained by direct purchase or by reference to published literature including, but not limited to, US 4623658A.
As used herein, the compound of formula IV has the formula
The compounds may be obtained by direct purchase or by reference to published literature including, but not limited to, WO2003082272a 1.
Here, the compound of formula VI has the formula
The compound can be obtained by direct purchase or by reference to published literature.
As used herein, the compound of formula VII has the formula
The compounds may be obtained by direct purchase or by reference to published literature including, but not limited to, WO2000042012a 1.
As used herein, the compound of formula VIII has the formula
The compounds may be obtained by direct purchase or by reference to published literature including, but not limited to, WO2000042012a 1.
As used herein, the compound of formula IX is of the formula
The compound can be obtained by direct purchase or by reference to published literature.
As used herein, a compound of formula X has the formula
The compound can be obtained by direct purchase or by reference to published literature including, but not limited to, CN 106674097A.
Here, the compound of formula XI is of formula
The compound can be obtained by direct purchase or ginsengPrepared according to published literature including, but not limited to, WO2016005874a 1.
In the present invention, unless otherwise indicated, a compound of formula I used in a "test article configuration" includes, but is not limited to, a newly prepared or stored compound of formula I or a pharmaceutical composition comprising a compound of formula I, preferably a newly prepared or stored compound of formula I.
In the present invention, the related substances are also expressed as impurities.
In the present invention, the appropriate amount means that the amount of each compound is within the detection limit or the quantitative limit of the HPLC thereof according to the purpose of the experiment.
In the present invention, "mL" means mL; "mg" means mg; "μ g" means micrograms; "min" means minutes;
in the present invention, "V: v: v "means volume ratio;
the method of the present invention is not limited to the 11 related substances, and any method of the present invention for separating and determining the compound of formula I and related substances falls within the scope of the present invention, and particularly, any compound of the compound of formula I and the compounds of formulae II to XII described in the present invention is separated and determined.
The invention aims to solve the technical problem of providing a high performance liquid chromatography analysis method capable of accurately determining the content of related substances of a test sample of a compound shown in the formula I.
The method provided by the invention can accurately determine the identified impurities (compounds shown in formulas II-XII) in the compound test sample shown in formula I, the separation degree of the main peak and the adjacent impurity peak is more than or equal to 1.5, and the separation degree between each impurity is more than or equal to 1.5, so that the qualitative and quantitative analysis of related substances of the test sample can be rapidly and accurately carried out, and the quality controllability of the product is ensured.
Drawings
FIG. 1 chromatogram of blank solution of example 2
FIG. 2 chromatogram of system adaptation solution of example 2
FIG. 3 chromatogram of test solution in example 2
FIG. 4 comparative example System suitability chromatogram
Detailed Description
The following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the scope of the present invention.
The reagents used in the invention: the compound of formula I test sample, the compound of formula IV reference sample, the compound of formula VI reference sample, the compound of formula VII reference sample, and the compound of formula X reference sample were purchased from Shanghai Boyue Biotech, Inc.; the compound of formula II control and the compound of formula III control were purchased from tokyo kekko limited; the compound of formula V control, the compound of formula VIII control, the compound of formula IX control, the compound of formula XI control, and the compound of formula XII control were purchased from haiyuekang pharmaceutical science co.
EXAMPLE 1 method for measuring substance
The test solution is prepared by dissolving appropriate amount of the product in diluent solvent [ acetonitrile-water-trifluoroacetic acid (V: V: V: 200: 800: 0.5) ] and diluting to obtain solution containing 0.5mg per 1 ml.
The reference solution is prepared by taking a regorafenib intermediate I reference substance and a proper amount of a compound of formula II, a compound of formula III, a compound of formula IV, a compound of formula V, a compound of formula VI, a compound of formula VII, a compound of formula VIII, a compound of formula IX, a compound of formula X, a compound of formula XI and a compound of formula XII, adding a diluting solvent to dissolve and dilute the reference solution to prepare a mixed solution containing about 5 mu g of regorafenib in 1 ml.
The system applicability solution takes a regorafenib intermediate I reference substance and a compound shown in a formula II, a compound shown in a formula III, a compound shown in a formula IV, a compound shown in a formula V, a compound shown in a formula VI, a compound shown in a formula VII, a compound shown in a formula VIII, a compound shown in a formula IX, a compound shown in a formula X, a compound shown in a formula XI and a reference substance shown in a formula XII in proper amount, and a diluting solvent is added for dissolving and diluting to prepare a mixed solution containing about 0.5mg of regorafenib intermediate I and 5 mu g of impurities in each 1 ml.
The chromatographic conditions are that Phenyl silane bonded silica gel is used as a filler Kromasil100-5Phenyl (4.6mm multiplied by 250mm, 5 mu m) is used as a chromatographic column; gradient elution was performed according to the following table using 20mmol/L potassium dihydrogen phosphate buffer-acetonitrile (95:5) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.9ml per minute; the column temperature was 25 ℃; the detection wavelength is 230 nm; the injector temperature was 4 ℃; the injection volume was 5. mu.l.
The measuring method comprises precisely measuring system applicability test solution, sample solution and reference solution, respectively injecting into liquid chromatograph, and recording chromatogram.
According to the chromatogram, calculating the content of each impurity in the sample by peak area through an external standard method:
and (3) calculating:
wherein: correction factor f ═ Cs/As;
cs is the concentration of each impurity of the reference solution, mu g/mL;
as-peak area of each impurity of the control solution;
at is the peak area of each impurity in the test solution;
n-volume of solvent of test solution, mL;
wt is the sample weighing and mg of the sample;
under the chromatographic conditions, two batches of VI compound samples are tested, wherein the content of each related substance is shown in Table 1,
TABLE 1 test article contents of the substances concerned
Example 2 specificity test
Diluent (b): acetonitrile-water-trifluoroacetic acid (V: V: V ═ 200: 800: 0.5)
Preparing an impurity reference substance stock solution: taking 20mg of each compound of the formula II-XII as reference, placing the reference in a 20ml volumetric flask, adding a diluent to dissolve and dilute the reference to obtain a solution containing 1.0mg of each impurity in 1ml, and using the solution as an impurity reference stock solution.
Compound of formula I control stock solution formulation: taking 20mg of a compound of the formula I as a reference substance, placing the reference substance into a 20ml volumetric flask, adding a diluent to dissolve and dilute the reference substance to prepare a solution containing 1.0mg of the compound of the formula I in each 1ml of the reference substance, and using the solution as a reference substance stock solution.
Preparing a positioning solution: precisely transferring 1ml of each impurity reference substance stock solution, placing into a 10ml measuring flask, diluting with diluent to obtain solution containing 50 μ g of each substance per 1ml, and using as positioning solution
Preparing a system applicability solution: taking about 25mg of the compound of formula I as a reference substance, precisely weighing, placing in a 50ml measuring flask, precisely removing 0.25ml of each of the reference substance stock solutions of the compound of formula II-XII, placing in a 50ml volumetric flask, dissolving and diluting with a diluent to obtain a solution containing about 0.5mg of the compound and 5 μ g of each related substance per 1ml, and using the solution as a system applicability solution.
Preparing a test solution: taking about 10mg of the compound of the formula I, precisely weighing, placing in a 20ml volumetric flask, adding a diluent to dissolve and dilute to prepare a solution containing about 0.5mg of the compound in each 1ml, and using the solution as a test solution.
The test process comprises the following steps: sampling blank solvent and above solution, recording chromatogram, and finding out the results shown in FIGS. 1-3.
The results show that: the blank has no interference, the separation degree of each related substance and the main peak is more than 1.5, and the system applicability is good.
Example 3 detection and quantitation limits
Solution preparation: the stock solutions of the reference substance of formula I and the impurity of formula II-XII in example 2 were precisely measured and diluted with appropriate amounts, and then diluted with acetonitrile-water-trifluoroacetic acid (V: 200: 800: 0.5) to a series of solutions, which were subjected to sample injection analysis under the chromatographic conditions described in example 1, and 6 quantification limiting solutions were prepared in parallel and tested, respectively, to examine the retention time RSD and peak area RSD, and confirm the precision of the method at the detection limit concentration. Determining the detection Limit (LOD) and the quantification Limit (LOQ) of the product by using a signal-to-noise ratio (S/N) method respectively: calculating by using the signal to noise ratio S/N which is more than or equal to 3:1, and taking the concentration of the corresponding test sample as a detection limit; the signal-to-noise ratio (S/N) method respectively determines the detection Limit (LOD) and the quantification Limit (LOQ) of the product, and the S/N is more than or equal to 9:1 and corresponds to the concentration of the test sample as the quantification limit.
The test process comprises the following steps: sampling blank solvent and the above solution, and recording chromatogram. The results are shown in tables 2 to 3.
TABLE 2 quantitative limit results
TABLE 3 Limit of detection results
The results show that: RSD of quantitative limit peak area of each impurity is not more than 10.0%, RSD of retention time is not more than 1.0%, LOQ and LOD are not more than 0.02% of concentration of a test sample, and sensitivity of each impurity meets requirements.
Example 4 linearity and Range
Solution preparation 1: 0.5ml of each of the reference substance stock solutions of the impurities of the formulae II to XII in example 2 and 1.0ml of the reference substance stock solution of the compound of the formula I were taken out of the flask and placed in a 20ml volumetric flask, and dissolved and diluted with a diluent to prepare a mixed solution containing about 50. mu.g of the compound of the formula I and 25. mu.g of each of the other related substances per 1ml, as a linear stock solution. Transferring 0.5ml of the stock solution into 20ml, 1ml of the stock solution into 20ml, 2ml of the stock solution into 25ml, 2ml of the stock solution into 20ml, 3ml of the stock solution into 25ml and 2ml of the stock solution into 10ml measuring flasks, diluting the stock solution with a diluent to a scale to obtain a series of linear solutions, and taking a quantitative limiting solution as a linear test solution.
Solution preparation 2: 1.5ml of the impurity control stock solution of the formula X, 3.0ml of each of the impurity control stock solutions of the formula XI and 1ml of the compound control stock solution of the formula I in example 2 were taken and placed in a 20ml measuring flask, and dissolved and diluted with a diluent to prepare a mixed solution containing about 50. mu.g of the compound of the formula I and 25. mu.g of each of the other related substances per 1ml, as a linear stock solution. Transferring 0.5ml of the stock solution into 20ml, 1ml of the stock solution into 20ml, 2ml of the stock solution into 25ml, 2ml of the stock solution into 20ml, 3ml of the stock solution into 25ml, 2ml of the stock solution into 10ml of measuring flask, diluting the stock solution with a diluent to a scale, and taking a quantitative limiting solution as a linear test solution.
The test method comprises the following steps: and precisely measuring the quantitative limiting solution and the linear test solution, respectively injecting the quantitative limiting solution and the linear test solution into a liquid chromatograph, and performing sample injection analysis, wherein the concentration is subjected to linear regression by peak area, and the result is shown in table 4.
TABLE 4 Linear and Range test results
Example 5 solution stability
0.5ml of each of the impurity reference stock solutions of the formulae II, III, IV, V, VI, VII, VIII, IX and XII in example 2, 1.5ml of the impurity reference stock solution of the formula X and 3ml of the impurity reference stock solution of the formula XI were placed in a 20ml volumetric flask, and dissolved and diluted with a diluent to prepare a solution containing about 75. mu.g of the compound of the formula X, 150. mu.g of the compound of the formula XI, the compound of the formula II, III, IV, V, VI, VII, VIII, IX and XII in each 1ml of the impurity mixed stock solutions. Taking about 10mg of the compound shown in the formula I, precisely weighing, placing in a 20ml volumetric flask, precisely weighing 2ml of impurity mixed stock solution, placing in the 20ml volumetric flask, dissolving and diluting with a diluent to obtain a solution containing about 0.5mg of the compound shown in the formula X, 7.5 μ g of the compound shown in the formula X, 15 μ g of the compound shown in the formula XI and 2.5 μ g of other related substances in each 1ml, and using the solution as a standard sample solution.
Preparing a test solution: weighing about 10mg of the compound of formula I, accurately weighing, placing in a 20ml volumetric flask, adding diluent to dissolve and dilute into a solution containing about 0.5mg of the compound per 1ml, and shaking up to obtain a test solution.
The test method comprises the following steps: adding the sample solution and the reference solution, respectively standing at room temperature for 0, 3, 6, 9, 12, 24, and 48 hr for sample introduction, calculating peak area RSD of main component and related substances, and recording chromatogram. The results are shown in tables 5 to 6.
TABLE 5 test article solution stability results
TABLE 6 stability results for solutions of spiked test samples
The results show that: the sample solution is placed at low temperature for 48 hours, the variation difference of each impurity is +/-0.03%, the detection conditions of related substances are basically consistent, no new impurity is added, and the stability is good; and placing the added standard sample solution at low temperature for 20 hours, wherein the difference between the content of each related substance and 0 hour is not more than 0.05%. The stability is good.
Example 6 precision experiments
6.1 precision of sample introduction
Solution preparation: the same standard test solution as in example 5 was prepared.
The test method comprises the following steps: and continuously injecting 6 needles, recording chromatograms, and calculating the RSD of the peak areas and retention times of the main components and related substances of the 6 needles. The results show that: RSD of the average peak area of each impurity is not more than 5.0%, RSD of retention time is not more than 1.0%, and sample introduction precision of an instrument is good.
6.2 repeatability
Preparing a test solution: the standard test solution was prepared in the same manner as in example 5, and 6 parts were prepared in parallel.
Preparing a reference solution: control solutions were prepared as in example 1.
The test method comprises the following steps: and sampling the added sample solution and the reference solution, recording the chromatogram, and calculating the RSD of the contents of the related substances in the 6 parts of mixed solution and the sample solution.
TABLE 7 results of repeated experiments
| Impurities | II | III | IV | V | VI | VIII | VIII | IX | X | XI | XII |
| RSD(％) | 0.9 | 4.6 | 1 | 0.9 | 1.3 | 1.3 | 1 | 1.2 | 1.1 | 0.8 | 1.2 |
The results show that: the RSD of each known impurity and the total impurity content in 6 parts of samples is not more than 10.0 percent, and the method has good repeatability.
6.3 intermediate precision
Solution preparation: the preparation method is the same with the repeatability.
The test method comprises the following steps: the sample preparation is carried out by preparing a standard sample solution and a reference solution according to a mode under a repeatability item, and carrying out intermediate precision tests by different analysts on different days.
TABLE 8 intermediate precision experimental results
| Name of impurity | II | III | IV | V | VI | VII | VIII | IX | X | XI | XII |
| RSD(％) | 1.2 | 5.6 | 1.2 | 1.4 | 1.9 | 1.5 | 1.4 | 0.8 | 1.6 | 1 | 1.6 |
As can be seen from table 7, the RSD (n ═ 12) of each compound content was 1% to 5.6%, indicating good intermediate precision of the method
Example 7 accuracy experiment
0.5ml of each of the II, III, IV, V, VI, VII, VIII, IX and XII control stock solutions of example 2, 1.5ml of the X control stock solution and 3ml of the XI control stock solution were placed in a 20ml volumetric flask and dissolved and diluted with a diluent to prepare a solution containing about 75. mu.g of X and 150. mu.g of XI and 25. mu.g of II, III, IV, V, VI, VII, VIII, IX and XII per 1ml of the solution as an impurity mixed stock solution. 3 parts are prepared in parallel.
Preparing a sample solution: taking about 10mg of regorafenib intermediate, precisely weighing, placing in a 20ml measuring flask, adding a diluent to dissolve and dilute to prepare a solution containing about 0.5mg of regorafenib intermediate in every 1ml, and taking the solution as a test solution.
Preparing a self control solution: precisely transferring 1ml of sample solution, placing the sample solution into a 100ml volumetric flask, adding a diluent to dilute to a constant volume to a scale, and shaking up to be used as a self-control solution.
Preparing 100% of reference substance solution: precisely transferring 2ml of the impurity mixed stock solution into a 20ml measuring flask, and adding a diluent to dilute the impurity mixed stock solution until each related substance is a solution with a limit concentration.
Preparing a recovery test solution: 1ml of impurity mixed reference substance stock solution is placed into 20ml, 2ml of impurity mixed reference substance stock solution is placed into 20ml, 3ml of impurity mixed reference substance stock solution is placed into 20ml of measuring flask, a proper amount of regorafenib raw material medicine is taken and placed into the measuring flask, a diluent is added for dissolution and dilution, so that about 0.5mg of regorafenib raw material medicine is contained in each 1ml of the regorafenib raw material medicine, each related substance is a solution with the concentration of 50%, 100% and 150% of the limit, and each concentration is 3 parts.
The test method comprises the following steps: respectively injecting blank solution, self-contrast solution, 100% contrast solution, sample solution and recovery rate sample solution, injecting into a liquid chromatograph, recording chromatogram, and calculating recovery rate by external standard method. The results are shown in tables 8 to 9.
TABLE 9 recovery results for compounds of formula IV
TABLE 10 average recovery and RSD of Compounds of formulae II-XII
| Name of impurity | II | III | IV | V | VI | VII | VIII | IX | X | XI | XII |
| Average recovery rate% | 102.4 | 103.6 | 100.9 | 101.7 | 100.4 | 100.6 | 101.8 | 102.6 | 107.3 | 105.7 | 97.3 |
| RSD(％) | 2.2 | 5.3 | 0.7 | 1.8 | 0.5 | 2.0 | 1.6 | 0.4 | 5.1 | 1.7 | 3.1 |
The results show that: the average value of the recovery rate of each impurity is between 80% and 120%, the RSD value is not more than 10.0%, and the method has good accuracy.
EXAMPLE 8 preparation of the Compound of formula V
| Name (R) | Molecular weight | Batch charging ratio | Feed amount | Molar weight of |
| II | 170.60 | 1 | 6g | 35.17mmol |
| III | 127.12 | 1.05 | 4.694g | 36.928mmol |
| EtOH | / | / | 60ml | / |
| HCL (liquid) | / | / | 3.5ml | / |
According to the feeding amount of the table, II (purchased from Shenghe Liang Zi) and III (purchased from Shenghe Liang Zi) and EtOH are added into a 100mL three-mouth reaction bottle in sequence, hydrochloric acid is added after nitrogen gas is replaced for three times, and the mixture is stirred, heated and refluxed and reacted for 2.5 hours. After TLC monitoring reaction is completed, the reaction solution is cooled to room temperature and then is decompressed and concentrated to be dry, 200ml of DCM and 200ml of purified water are added, liquid separation is carried out by stirring, an aqueous phase is collected, the aqueous phase is extracted and separated by 100ml of DCM, and the aqueous phase is collected. And adjusting the pH of the water phase to 8-9 by using a saturated sodium bicarbonate water solution, and crystallizing for 1h at 10-20 ℃ after a large amount of solids are precipitated. Filtering and drying to obtain solid 5.1g, namely the compound of the formula V. 1 H-NMR(500MHz,DMSO-d6)δ(ppm)2.76(d，J＝6.0Hz，3H)，6.65-6.70(m，3H)，7.15(t，J＝12.0Hz，1H)，7.23(s，1H)，8.12(d，J＝6.0Hz，1H)，8.52(s，1H)，8.58(d，J＝6.0Hz，1H)，9.91(s，1H)。
Example 9 preparation of a Compound of formula XII
| Name (R) | Molecular weight | Batch charging ratio | Feed amount | Molar weight of |
| XI | 395.39 | 1 | 2g | 5.06mmol |
| II | 170.60 | 1.1 | 0.95g | 5.56mmol |
| t-BuOK | 112.21 | 1.1 | 0.625g | 5.56mmol |
| THF | / | N/A | 10ml | / |
| NMP | / | N/A | 20ml | / |
| Potassium carbonate | 138.21 | 0.6 | 0.42g | 3.036mmol |
Adding XI (self-made in reference documents), II (self-made in Hedgeon and Biotechnology), THF and NMP into a 100mL three-mouth reaction bottle in sequence according to the feeding amount in the table, replacing nitrogen for three times, adding potassium tert-butoxide and potassium carbonate, stirring and heating to 90-100 ℃, and carrying out reflux reaction for 9 hours. And (3) after TLC monitoring reaction is finished, cooling to 50 ℃, concentrating under reduced pressure to remove THF, then controlling the temperature to be 15-20 ℃, dropwise adding 25ml of purified water, stirring for crystallization for 16 hours, filtering, pulping the solid with EA: EtOH (1: 1), filtering, and drying. To obtain 700mg of solid, namely the compound shown in the formula XII. 1 H-NMR(500MHz,DMSO-d6)δ(ppm)2.80-2.83(m，9H)，7.23-7.24(m，3H)，7.31(s，1H)，7.53(d，J＝10.0Hz，1H)，7.62-7.64(m，4H)，8.51-8.52(m，2H)，8.60-8.61(m，1H)，8.80-8.83(m，3H)。
Comparative examples
Solution preparation: the test solution, the test solution and the control solution were prepared in the same manner as in example 1.
Chromatographic conditions a column using octadecylsilane bonded silica gel as a filler [ Waters Symmetry C18(250 mm. times.4.6 mm, 5 μm) ]; gradient elution was performed according to the following table using 20mmol/L potassium dihydrogen phosphate buffer-acetonitrile (95:5) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.9ml per minute; the column temperature was 25 ℃; the detection wavelength is 230 nm; the injector temperature was 4 ℃; the injection volume was 5. mu.l.
The measurement method comprises precisely measuring system applicability test solution, sample solution and control solution, respectively injecting into liquid chromatograph, and recording chromatogram, as shown in FIG. 4.
The results show that the compound of formula IV and the compound of formula V show peak coincidence, and the compound of formula X and the compound of formula VII show peak coincidence. It was shown that the separation effect of the column using octadecylsilane chemically bonded silica as a filler was inferior to that of the column using phenylsilane chemically bonded silica as a filler.
Claims (10)
1. A regorafenib intermediate related substance analysis method is characterized by comprising the following steps: the method is a high performance liquid chromatography, and the method adopts a reversed phase chromatographic column, takes a mixed solution of potassium dihydrogen phosphate buffer solution and acetonitrile as an eluent, and elutes according to isocratic or gradient, wherein the structural formula of a regorafenib intermediate is shown in the specification
2. The assay of claim 1, wherein: the method is a high performance liquid chromatography, and the method adopts a reversed phase chromatographic column, and takes a mobile phase A and/or a mobile phase B as an eluent, wherein the mobile phase A is potassium dihydrogen phosphate buffer solution-acetonitrile, and the mobile phase B is acetonitrile; in the mobile phase A, the volume ratio of the potassium dihydrogen phosphate buffer solution to the acetonitrile is 95: 5.
3. the assay of claim 2, wherein the mobile phase a and mobile phase B are subjected to a gradient elution according to the following table:
in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of the mobile phase A is the percentage of the volume of the mobile phase A to the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the volume of the mobile phase B to the total volume of the eluent.
4. The assay method according to claim 1, wherein the concentration of the potassium dihydrogen phosphate buffer is 0.005mol/L to 0.2 mol/L; preferably 0.02mol/L to 0.04 mol/L; more preferably 0.02 mol/L.
5. The analytical method of claim 1, wherein the flow rate of the eluent is 0.5 to 2 mL/min; preferably 0.5-1 mL/min; more preferably 0.9 mL/min.
6. The analytical method of claim 1, wherein the reverse phase chromatographic column uses a non-polar stationary phase as packing; phenyl silane bonded silica gel is preferably used as a filler; more preferably, the reverse phase chromatography column is Kromasil100-5Phenyl, which is 250mm by 4.6mm, 5 μm in size.
7. The analytical method of claim 1, which is performed on a high performance liquid chromatograph using a diode array detector, an ultraviolet detector, a differential refractive detector, an electrospray detector, or an evaporative light scattering detector; preferably, the analysis method is performed on a high performance liquid chromatograph, and a diode array detector or an ultraviolet detector is adopted; further preferably, the analysis method is carried out on a high performance liquid chromatograph, an ultraviolet detector is adopted, and the detection wavelength is 195 nm-400 nm; more preferably 200nm to 322 nm; most preferably 230 nm.
8. The analytical method of claim 1, wherein the reverse phase chromatography column has a column temperature of 20 to 55 ℃; the preferable column temperature is 20-30 ℃; more preferably, the column temperature is 25 ℃.
9. The assay of claim 1, wherein:
the analysis method is carried out on a high performance liquid chromatograph; the method adopts a reverse phase chromatographic column, and the reverse phase chromatographic column adopts phenyl silane bonded silica gel as a filler;
the analysis method adopts an ultraviolet detector, and the detection wavelength is 230 nm;
the analysis method adopts a reversed phase chromatographic column, and the column temperature is 25 ℃;
the analysis method uses a mobile phase A and/or a mobile phase B as an eluent, wherein the mobile phase A is potassium dihydrogen phosphate buffer solution-acetonitrile, the mobile phase B is acetonitrile, the potassium dihydrogen phosphate buffer solution is 0.02mol/L potassium dihydrogen phosphate aqueous solution, and the volume ratio of the potassium dihydrogen phosphate aqueous solution to the acetonitrile in the mobile phase A is 95: 5;
gradient elution was performed according to the following procedure:
in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of the mobile phase A is the percentage of the volume of the mobile phase A in the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the volume of the mobile phase B in the total volume of the eluent;
the flow rate of the eluate was 0.9 mL/min.
10. A compound of formula V, XII for use in preparing an impurity control for regorafenib intermediate related substance assays, wherein the compound of formula V, XII has the structure:
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