CN115774062A - 3, 5-dichlorobenzoic acid related substance analysis method - Google Patents
3, 5-dichlorobenzoic acid related substance analysis method Download PDFInfo
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- 239000000126 substance Substances 0.000 title claims abstract description 24
- CXKCZFDUOYMOOP-UHFFFAOYSA-N 3,5-dichlorobenzoic acid Chemical compound OC(=O)C1=CC(Cl)=CC(Cl)=C1 CXKCZFDUOYMOOP-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 238000004458 analytical method Methods 0.000 title claims description 24
- 239000012535 impurity Substances 0.000 claims abstract description 142
- 150000001875 compounds Chemical class 0.000 claims abstract description 65
- 238000000034 method Methods 0.000 claims abstract description 35
- 238000001514 detection method Methods 0.000 claims abstract description 18
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims abstract description 16
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 16
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
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- TXEIIPDJKFWEEC-UHFFFAOYSA-N tafamidis Chemical compound O1C2=CC(C(=O)O)=CC=C2N=C1C1=CC(Cl)=CC(Cl)=C1 TXEIIPDJKFWEEC-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a method for analyzing related substances of 3, 5-dichlorobenzoic acid, which adopts a reversed-phase high performance liquid chromatography to separate and measure a compound shown in a formula I and an impurity A, a compound shown in an impurity B, a compound shown in an impurity C, a compound shown in an impurity D, a compound shown in an impurity E and a compound shown in an impurity F. The method has the advantages that the solvent does not interfere with impurity detection, the method has good specificity, data support and reference are provided for the preparation of the quality standard of the 2- (3, 5-dichlorophenyl) -6-benzoxazole formic acid and the screening of the preparation process, and the method has important practical significance in the aspects of production and quality control.
Description
Technical Field
The invention relates to the field of pharmaceutical analysis, in particular to an analysis method of 3, 5-dichlorobenzoic acid related substances.
Background
3, 5-Dichlorobenzoic acid is an important starting material in the field of chemical medicine, and has a molecular formula of C 7 H 4 Cl 2 O 2 Molecular weight: 191.01, structural formula:
the potential impurities of the 3, 5-dichlorobenzoic acid related substances can be researched and analyzed in the production process of the 3, 5-dichlorobenzoic acid related substances through detection and analysis, the quality control of the related substances of reactants in the subsequent pharmaceutical process is effectively ensured, the generation of side reactions and the generation of impurities are reduced, data support and reference are provided for the preparation of the quality standard of the 2- (3, 5-dichlorophenyl) -6-benzoxazole carboxylic acid and the screening of the preparation process, and the method has important practical significance in the aspects of production and quality control. The following impurities may be involved in the 3, 5-dichlorobenzoic acid feed, which may have an effect on product quality.
Disclosure of Invention
The invention provides an analysis method of 3, 5-dichlorobenzoic acid related substances shown as a formula I:
in order to realize the purpose, the technical scheme of the invention is as follows: a method for analyzing 3, 5-dichlorobenzoic acid related substances is characterized by comprising the following steps: the method is a high performance liquid chromatography, and adopts a reversed phase chromatographic column and takes a phosphoric acid solution as a mobile phase A; acetonitrile is used as a mobile phase B, and elution is carried out according to isocratic or gradient.
In some embodiments, the gradient elution is performed with phosphoric acid solution as mobile phase a and acetonitrile as mobile phase B according to the following table:
in the 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 mobile phase A to the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the mobile phase B to the total volume of the eluent.
In some exemplary embodiments, the gradient elution is performed according to the following table:
in some embodiments, the concentration of the phosphoric acid solution is 0.05 to 0.5%, preferably 0.05 to 0.2%, more preferably 0.09 to 0.11%, and most preferably 0.1%.
In some embodiments, the eluent has a flow rate of 0.8 to 1.2ml/min; in some typical embodiments, the eluent has a flow rate of 0.9 to 1.1ml/min; in some more typical embodiments, the flow rate of the eluent is 1ml/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 a Waters column
T3, 250mm × 4.6mm in specification, 5 μm.
In some embodiments, the analytical method is performed on a high performance liquid chromatograph, employing a diode array detector, an ultraviolet detector, a differential refractive detector, an electrospray detector, or an evaporative light scattering detector; in some exemplary embodiments, a diode array detector or ultraviolet detector is employed; in some more typical embodiments, an ultraviolet detector is employed.
In some embodiments, the detection wavelength is between 195nm and 400nm; preferably 200nm to 220nm; more preferably 210nm.
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 30 to 40 ℃; in some more typical embodiments, the column temperature of the reverse phase chromatography column is 35 ℃.
In another aspect, the present invention provides a method for analyzing a 3, 5-dichlorobenzoic acid-related substance, comprising:
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 210nm;
the column temperature of the analysis method is 35 ℃;
the analysis method comprises the steps of taking a mobile phase A and a mobile phase B as eluents, wherein the mobile phase A is a phosphoric acid solution, the mobile phase B is acetonitrile, and the phosphoric acid solution is 0.1%;
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 1.0ml/min.
Respectively injecting a test solution, a reference solution and a system applicability solution of the compound of the formula I;
the reference solution comprises a compound of formula I and one or more of impurity A compound, impurity B compound, impurity C compound, impurity D compound, impurity E compound and impurity F compound;
the system suitability solution comprises a compound of formula I and one or a mixture of more than two of an impurity A compound, an impurity B compound, an impurity C compound, an impurity D compound, an impurity E compound and an impurity F compound;
the contents of the compound I, the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F are calculated by a main component self-comparison method.
In certain embodiments, the present invention provides a method for analyzing a substance of formula I, comprising the steps of:
(1) Preparing a compound I positioning solution: taking a proper amount of a reference substance shown in the formula I, dissolving and diluting the reference substance by using a mixed solution of acetonitrile-water (50);
optionally, comprising step (2) impurity a localization solution formulation: taking a proper amount of an impurity A reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
optionally, comprising step (3) impurity B localization solution preparation: taking a proper amount of an impurity B reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
optionally, comprising step (4) impurity C localization solution preparation: taking a proper amount of an impurity C reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
optionally, comprising step (5) impurity D localization solution formulation: taking a proper amount of an impurity D reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
optionally, comprising step (6) impurity E localization solution formulation: taking a proper amount of an impurity E reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
optionally, comprising step (7) impurity F localization solution formulation: taking a proper amount of an impurity E reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50); and
preparing a system applicability solution comprising the following steps (8): taking a proper amount of each compound I and impurities, precisely weighing, dissolving and diluting with a mixed solution of acetonitrile-water (50);
(9) Preparing a test solution: taking a proper amount of a compound I test sample, precisely weighing, dissolving with a mixed solution of acetonitrile-water (50);
(10) Preparing a reference solution: precisely measuring a proper amount of a compound of the formula I as a reference substance solution, and diluting the reference substance solution to a solution containing 1 mu g of the compound per 1ml by using an acetonitrile-water (50);
(11) Liquid phase conditions: using a Waters Atlantis T3 octadecylsilane chemically bonded silica chromatographic column with the specification of 4.6mm multiplied by 250mm and 5 mu m, wherein the column temperature is 35 ℃; the detector is an ultraviolet absorption detector, and the detection wavelength is 210nm; the sample injector temperature is 35 ℃; the mobile phase A is 0.1 percent phosphoric acid solution; the mobile phase B is acetonitrile, the flow rate of the mobile phase is 1.0ml/min, and the elution is carried out according to the following gradient:
(12) And (3) sample determination: injecting a proper amount of a positioning solution of the compound I, a positioning solution of each impurity, a proper amount of a system applicability solution, a proper amount of a test solution and a proper amount of a reference solution into a liquid chromatograph, and recording chromatograms of the solutions;
(13) And (3) calculating the content: based on each spectrogram, self-contrast method of principal component with or without correction factor
Calculating the content of each impurity in the sample:
wherein At is the peak area of impurities in the chromatogram of the test solution; a is the peak area of the main peak in the control solution, namely the peak area of the compound I; f is a correction factor; m is the dilution multiple.
Those skilled in the art will readily understand that the compounds used for preparing the positioning solution in steps (2) to (7) have a corresponding relationship with the impurities in step (8) according to different purposes; specifically, for example, when the content of the impurity a in the test sample is determined, the method at least includes the step (2), and the impurity of the step (8) at least includes the impurity a; for another example, when the contents of the impurities B and C in the sample are simultaneously measured, the method at least comprises the steps (3) and (4), and the related substance in the step (8) at least comprises the impurities B and C.
In some embodiments, if there is an impurity peak in the chromatogram of the test solution, the content of impurity a is not higher than 0.5%, preferably not higher than 0.2%; the content of impurity B is not higher than 0.5%, preferably not higher than 0.2%; the content of impurity C is not higher than 0.5%, preferably not higher than 0.2%; the content of the impurity D is not higher than 0.5%, preferably not higher than 0.2%; the content of the impurity E is not higher than 0.5%, preferably not higher than 0.2%; the total content of each impurity is not more than 2.0%, preferably not more than 0.5%.
Herein, formula I, impurity a, impurity B, impurity C, impurity D, impurity E and impurity F can all be obtained by direct purchase or preparation according to published literature.
Herein, unless otherwise specified, compound I formulated in "test article solutions" includes, but is not limited to, freshly prepared or stored drug substances and formulation compositions comprising compound I.
In this context, substances of interest are also denoted as impurities.
The concentration of the solution is positioned to be 100 mu g/ml-1mg/ml; preferably 100. Mu.g/ml to 10. Mu.g/ml; more preferably 1. Mu.g/ml.
System applicability the concentration of compound I in solution was: 1 mu g/ml-10mg/ml; preferably 0.1mg/ml to 1mg/ml; more preferably 0.5mg/ml.
The concentration of impurity A, impurity B, impurity C, impurity D or impurity E in the system applicability solution is as follows: 100 mu g/ml to 0.5mg/ml; preferably 0.2. Mu.g/ml to 10. Mu.g/ml; more preferably 1. Mu.g/ml.
The concentration of compound I in the test solution is 0.01mg/ml to 10mg/ml, preferably 0.1mg/ml to 1mg/ml, and more preferably 0.5mg/ml.
In the control solution, the concentration of the compound I is 0.1 to 20 mu g/ml; preferably 0.2 to 2 mug/ml; more preferably 1. Mu.g/ml.
The correction factor can be calculated by precisely weighing appropriate amounts of the main component (compound I) reference substance and the impurity reference substance to respectively prepare solutions with different concentrations, detecting by HPLC, drawing a regression curve of the main component concentration and the impurity concentration to the peak area thereof, and calculating the correction factor according to the ratio of the main component regression line slope to the impurity regression line slope.
Preparation of 0.1% phosphoric acid solution: accurately weighing 1ml of phosphoric acid, and adding water to 1000ml to obtain the product.
The invention provides an analysis method of related substances of a compound I, which can realize the complete separation of all impurities in the compound I, and the separation degree of a main component and each impurity is more than 1.5, and the separation degree of each impurity is more than 1.5. The method of the invention can be used for quickly and accurately carrying out quantitative analysis on related substances of the compound I with high sensitivity, thereby ensuring the quality controllability of the product.
Drawings
FIG. 1: example 1 blank solvent chromatogram;
FIG. 2: example 1 chromatogram for system suitability solution assay;
FIG. 3: example 1 chromatogram for the assay of a test solution of compound I;
Detailed Description
The following examples will provide those skilled in the art with a more complete understanding of the present invention, but are not intended to limit the scope of the present invention to the examples. The method of the present invention is not limited to the above 6 related substances, and any method of separating and measuring compound I and related substances by the method of the present invention falls within the scope of the present invention, and any combination of the impurities A, B, C, D, E, and F in compound I and the above impurities by the method of the present invention falls within the scope of the present invention.
Example 1
A chromatographic column: waters Atlantis T3 (4.6 mm. Times.250mm, 5 μm)
Detection wavelength: 210nm
Flow rate of mobile phase: 1.0ml/min
Column temperature: 35 deg.C
Sample injection amount: 10 μ l
Mobile phase: the mobile phase A is 0.1 percent phosphoric acid solution; mobile phase B was acetonitrile and elution gradient was as follows:
solvent: acetonitrile-water (50
Stock solutions of various impurities: taking a proper amount of an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D, an impurity E reference substance and an impurity F reference substance, precisely weighing, respectively placing into different measuring bottles, adding a solvent to dissolve and dilute to prepare a solution containing 0.4mg of the impurity A reference substance, the impurity B reference substance, the impurity C reference substance, the impurity D, the impurity E reference substance and the impurity F reference substance in each 1ml, and taking the solution as a stock solution of each impurity reference substance.
Preparing an impurity positioning solution: respectively taking 1ml of each impurity stock solution, respectively placing the impurity stock solutions in 20ml measuring flasks, diluting the impurity stock solutions to the scales by using a solvent, shaking up, respectively taking 1ml of each impurity stock solution, respectively placing the impurity stock solutions in 20ml measuring flasks, diluting the impurity stock solutions to the scales by using the solvent, and shaking up to be used as positioning solutions of the impurities.
Preparing a system applicability solution: accurately measuring each impurity reference substance and compound I reference substance respectively, and diluting with solvent to obtain a mixed solution containing about 0.5mg of compound I, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F, 1 μ g per 1 mL.
Control solution: taking a proper amount of a compound I reference substance, adding a solvent to dissolve and dilute the compound I reference substance to prepare a solution containing about 1 mu g of the compound I per 1ml, and using the solution as a reference substance solution.
Preparing a test solution: taking a proper amount of a compound I reference substance, dissolving with a solvent, and diluting to prepare a solution containing about 0.5mg of the compound I in each 1 ml.
And (3) experimental determination: taking blank solution, positioning solutions and reference solution 10 μ l each, and injecting into high performance liquid chromatograph, wherein the chromatogram of the blank solution is shown in figure 1.
Injecting 10 μ l of the system applicability solution into high performance liquid chromatograph, recording chromatogram as figure 2,
taking 10 μ l of test solution of compound I, injecting into high performance liquid chromatograph, recording chromatogram shown in figure 3, and calculating according to self-control method of adding correction factor, wherein the content of each related substance is shown in table 1.
TABLE 1 test article contents of the substances concerned
| Name of impurity | Content (%) |
| Impurity A | 0.01 |
| Impurity B | Undetected |
| Impurity C | 0.09 |
| Impurity D | Not detected out |
| Impurity E | Not detected out |
| Impurity F | Not detected out |
| Other single impurities | 0.03 |
| Total amount of impurities | 0.14 |
The experimental results are as follows: the blank solution has no interference to the measurement, the separation degree of each related substance and the main peak is more than 1.5, and the system applicability is good. The content of each impurity is not more than 0.1%, the content of other single impurities is not more than 0.05%, and the total content of the impurities is not more than 0.2%.
Example 2 detection and quantitation limits
Solution preparation: precisely measuring appropriate amounts of the reference substance stock solution of the formula I and the impurity stock solutions A-F in example 1, gradually diluting the solutions with acetonitrile-water (50) to form a series of solutions, carrying out sample injection analysis under the chromatographic conditions described in example 1, preparing 6 parts of quantitative limiting solutions in parallel, testing the solutions respectively, and inspecting the RSD of retention time and the RSD of peak area to confirm the precision of the method under 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 taking the signal-to-noise ratio S/N to be more than or equal to 3 and taking the corresponding concentration of the test sample as a detection limit; the S/N ratio is calculated according to the ratio of S/N to S/N being more than or equal to 10, the concentration of the corresponding test sample is taken as a quantitative limit, and the result is shown in tables 2 and 3.
Table 2: quantitative limit result
Table 3: limit of detection result
The results show that: RSD of quantitative limit peak area of each impurity is not more than 10.0%, and RSD of retention time is not more than 1.0%; the quantitative limit concentration is not more than 50% of the impurity limit concentration.
Example 3 linearity and Range
Solvent: acetonitrile-water (50
Stock solutions of each impurity:
taking a proper amount of a compound reference substance of the formula I, an impurity A reference substance, an impurity B reference substance, an impurity C reference substance, an impurity D reference substance, an impurity E reference substance and an impurity F reference substance, precisely weighing, respectively placing into different measuring bottles, adding a solvent to dissolve and dilute to prepare a solution containing 0.4mg of the compound of the formula I, the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F in each 1ml, and taking another quantitative limiting solution as a linear test solution.
Mixed impurity stock solution:
precisely measuring appropriate amount of each impurity stock solution, diluting with solvent to obtain 20 μ g solution containing compound I reference substance, impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance and impurity F reference substance per 1ml, diluting with diluent to scale, and taking quantitative limit solution as linear test solution. Linear solution formulation is shown in table 4:
table 4: linear solution preparation table
The results show that: the correlation coefficient r is not less than 0.998; the Y-axis intercept is within 25% of the 100% response value; response factor RSD is no greater than 10%;
EXAMPLE 4 durability test
The durability of the detection method was examined by changing the conditions of flow rate, column temperature, phosphoric acid concentration, organic phase ratio and the like on the basis of the conditions of the detection chromatography in example 1. Solution preparation and measurement method referring to example 1, the chromatographic conditions and results are shown in tables 5 and 6:
TABLE 5 durability chromatography conditions
Table 6: durability chromatography results:
and (4) conclusion: under different chromatographic conditions, the peak of the compound I and the adjacent chromatographic peaks can achieve baseline separation, the separation degree between the impurity peaks is more than or equal to 1.5, and the method has good durability.
Example 5 accuracy
Solvent: acetonitrile-water (50.
Stock solutions of various impurities: each impurity stock solution was prepared according to example 1.
Mixed impurity stock solution: a proper amount of each impurity stock solution is precisely measured respectively, and diluted by a solvent to prepare a solution containing 20 mu g of impurities A, B, C, D, E and F in each 1ml of the solution to serve as a mixed impurity stock solution.
System applicability solution: a system suitability solution was prepared as in example 1.
Control solution: taking a proper amount of a reference substance of the formula I, adding a solvent to dissolve and dilute the reference substance to prepare a solution containing about 1 mu g of reference substance per 1ml, and using the solution as a reference substance solution.
Test solution: about 10mg of the compound of formula I is taken, precisely weighed, placed in a 20ml measuring flask, dissolved with a solvent and diluted to obtain a solution containing about 0.5mg of the compound of formula I per 1ml, as a test solution.
Recovery rate solution: taking a compound sample of the formula I, weighing about 10mg precisely, placing the sample into a 20ml measuring flask, adding a proper amount of solvent to dissolve the compound sample, precisely adding a proper amount of mixed impurity stock solution, diluting the solution to a scale by using the solvent, and shaking up the solution to obtain a recovery solution. 3 parts of each concentration level and 6 parts of each 100 percent concentration level are prepared in parallel, and a repeatability test is carried out at the same time, the preparation method is shown in a table 7, and the experimental result is shown in a table 8.
Table 7: recovery rate solution preparation method
| Level of | Sample (mg) | Volume of control stock solution (ml) | Volumetric flask (ml) |
| 50％ | 10 | 0.5 | 20 |
| 100％ | 10 | 1.0 | 20 |
| 150％ | 10 | 1.5 | 20 |
Table 8: average recovery of impurities A-F and RSD
The experimental results are as follows: the recovery rate under each concentration is between 85 and 102 percent; the RSD of the recovery rate is not more than 10.0 percent.
Example 6 precision experiments
6.1 precision of sample introduction
Control solution: a control solution was prepared as in example 1.
The test method comprises the following steps: and continuously feeding samples into 6 needles, recording chromatograms, and calculating the RSD of the peak areas and retention times of the main components and all related substances of the 6 needles.
The results show that: RSD of the average peak area of each impurity is not more than 2.0%, 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: a standard sample solution was prepared in the same manner as in example 5, and 6 parts were prepared in parallel.
Preparing a reference substance solution: a control solution was prepared as in example 5.
The test method comprises the following steps: and (3) sampling the added standard test sample solution and the reference solution, recording the chromatogram, calculating the RSD of the contents of the related substances in the 6 parts of mixed solution and the test sample solution, and obtaining the experimental result shown in the table 9.
Table 9: results of repeatability experiments
The results show that: in 6 parts of the added standard sample solution, the RSD of each known impurity detection amount is less than or equal to 10 percent, the range difference of other single impurity detection amounts is less than or equal to 0.05 percent, and the RSD of the total amount of impurities is less than or equal to 10.0 percent.
6.3 intermediate precision
Solution preparation: the standard test solution was prepared as in example 5.
The test method comprises the following steps:
system applicability solution: a system compatible solution was prepared as in example 1.
Adding a standard test solution: the standard test sample solution was prepared as in example 5.
Control solution: control solutions were prepared as in example 5.
Different experimenters measure with different types of instruments at different times to perform intermediate precision tests, and the test results are shown in Table 10.
Table 10: results of intermediate precision experiment
The results show that: combining with a repeatability test, the RSD of the detected amount of each known impurity is less than or equal to 10 percent, the range of the detected amount of other single impurities is less than or equal to 0.05 percent, and the RSD of the total amount of the impurities is less than or equal to 10.0 percent in 12 parts of test solution.
Claims (10)
1. A method for analyzing 3, 5-dichlorobenzoic acid related substances is characterized by comprising the following steps: the method is a high performance liquid chromatography, and adopts a reversed phase chromatographic column and takes phosphoric acid solution as a mobile phase A; acetonitrile is used as a mobile phase B, and elution is carried out according to isocratic or gradient.
2. The assay of claim 1, wherein: taking phosphoric acid solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table:
in the 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 mobile phase A to the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the mobile phase B to the total volume of the eluent.
3. The analysis method according to claim 2, wherein a phosphoric acid solution is used as a mobile phase A; acetonitrile as mobile phase B, gradient elution was performed according to the following table:
in the 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 mobile phase A to the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the mobile phase B to the total volume of the eluent.
4. The assay of claim 1, wherein the concentration of the phosphoric acid solution is between 0.05 and 0.5%, preferably between 0.05 and 0.2%, more preferably between 0.09 and 0.11%, and most preferably 0.1%.
5. The analytical method of claim 1, wherein the flow rate of the eluent is 0.8 to 1.2ml/min; preferably 0.9-1.1 mL/min; more preferably 1ml/min.
6. An analytical method according to claim 1, wherein the reversed phase chromatographic column employs 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 Waters TLAntiiss T3, which has a specification of 250 mm. Times.4.6 mm,5 μm.
7. The analytical method of claim 1, which is performed on a high performance liquid chromatograph using a diode array detector, a uv 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 220nm; most preferably 210nm.
8. The analytical method of claim 1, wherein the reverse phase chromatographic column has a column temperature of 20 to 55 ℃; the column temperature is preferably 30-40 ℃; more preferably, the column temperature is 35 ℃.
9. The assay of claim 1, wherein: the analysis method is carried out on a high performance liquid chromatograph; the method adopts a reversed-phase chromatographic column, and the reversed-phase chromatographic column adopts phenyl silane bonded silica gel as a filler;
the analysis method adopts an ultraviolet detector, and the detection wavelength is 210nm;
the analysis method adopts a reversed phase chromatographic column, and the column temperature is 35 ℃;
the analysis method takes a mobile phase A and a mobile phase B as eluents, wherein the mobile phase A is a phosphoric acid solution, the mobile phase B is acetonitrile, and the phosphoric acid solution is 0.1%.
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 1.0ml/min.
10. The analytical method of claim 1, comprising the steps of:
(1) Preparing a compound I positioning solution: taking a proper amount of a reference substance shown in the formula I, dissolving and diluting the reference substance by using a mixed solution of acetonitrile-water (50);
wherein, the structural formula of the compound of formula I is:
optionally, comprising step (2) impurity a localization solution formulation: taking a proper amount of an impurity A reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
wherein, the structural formula of the impurity A compound is as follows:
optionally, comprising step (3) impurity B localization solution preparation: taking a proper amount of an impurity B reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
wherein, the structural formula of the impurity B compound is as follows:
optionally, comprising step (4) impurity C localization solution formulation: taking a proper amount of an impurity C reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
wherein, the structural formula of the impurity C compound is as follows:
optionally, comprising step (5) impurity D localization solution preparation: taking a proper amount of an impurity D reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
wherein, the structural formula of the impurity D compound is as follows:
optionally, comprising step (6) impurity E localization solution formulation: taking a proper amount of an impurity E reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
wherein, the structural formula of the impurity E compound is as follows:
optionally, comprising step (7) impurity F localization solution formulation: taking a proper amount of an impurity E reference substance, precisely weighing, dissolving and diluting with an acetonitrile-water (50); and
wherein, the structural formula of the impurity F compound is as follows:
preparing a system applicability solution comprising the following steps (8): taking a proper amount of each compound I and impurities, precisely weighing, dissolving and diluting with a mixed solution of acetonitrile-water (50);
(9) Preparing a test solution: taking a proper amount of a compound I test sample, precisely weighing, dissolving and diluting with an acetonitrile-water (50);
(10) Preparing a reference substance solution: precisely weighing a proper amount of a compound of formula I as a reference substance, diluting the reference substance with a mixed solution of acetonitrile-water (50);
(11) Liquid phase conditions: using a Waters Santlantis T3, an octadecylsilane chemically bonded silica chromatographic column with the specification of 4.6mm multiplied by 250mm and 5 mu m, and the column temperature is 35 ℃; the detector is an ultraviolet absorption detector, and the detection wavelength is 210nm; the injector temperature was 35 ℃; the mobile phase A is 0.1 percent phosphoric acid solution; the mobile phase B is acetonitrile, the flow rate of the mobile phase is 1.0ml/min, and the elution is carried out according to the following gradient:
(12) And (3) sample determination: injecting a proper amount of the positioning solution of the compound I, the positioning solution of each impurity, the system applicability solution, the test solution and the reference solution into a liquid chromatograph respectively, and recording the chromatogram of each solution;
(13) And (3) calculating the content: based on each chromatogram, by principal component self-comparison method with or without correction factor
Calculating the content of each impurity in the sample:
in the formula, at is the peak area of impurities in the chromatogram of the test solution; a is the peak area of the main peak in the control solution, namely the peak area of the compound I; f is a correction factor; m is the dilution multiple.
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