CN113390983B - Detection method for simultaneously determining 3 impurities in Voranolan fumarate - Google Patents
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Abstract
The invention belongs to the technical field of drug analysis and detection, and particularly relates to a detection method for simultaneously determining 3 impurities in Voranolan fumarate. The method can simultaneously determine 3 impurities of pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate in Voranolan fumarate under the condition of one chromatogram, has good separation degree of each chromatogram peak, high sensitivity, high recovery rate, high precision and stability, good reproducibility and high accuracy, can be used for monitoring impurities in the production process of Voranolan fumarate, and is beneficial to controlling and improving the quality of Voranolan fumarate; meanwhile, the method is simple to operate, short in detection time and very suitable for quality monitoring of the bulk drugs in large-scale industrial production.
Description
Technical Field
The invention belongs to the technical field of drug analysis and detection. More particularly, the invention relates to a detection method for simultaneously determining 3 impurities in Voranolan fumarate.
Background
Vonopalafenan fumarate (1- [5- (2-fluorophenyl) -1- (pyridin-3-ylsulfonyl) -1H-pyrrol-3-yl ] -N-methylmethanemethanamine monofumarate) is a novel proton pump inhibitor, and is mainly used for the treatment of acid-related gastrointestinal diseases, such as gastric ulcer, gastroesophageal reflux disease, etc. Pyridine-3-sulfonyl chloride is a key starting material for synthesizing Voranolan fumarate, is also a strong oxidant, has active chemical property, is easy to hydrolyze to generate pyridine-3-sulfonic acid, and simultaneously, the pyridine-3-sulfonyl chloride can also react with methanol and ethanol solvents used in the synthesis process to generate pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate which are potential genotoxic impurities; on the other hand, vorexan fumarate itself can also be degraded to give pyridine-3-sulfonic acid.
The impurities of the pyridine-3-sulfonic acid, the pyridine-3-methyl sulfonate and the pyridine-3-ethyl sulfonate which exist in the preparation process of the vorexan fumarate can be brought into a finished product, and the product has no treatment effect or influences the stability and the curative effect of the medicament, even has genetic toxicity, directly causes DNA damage, causes DNA mutation, and further causes cancer, and has potential safety hazard. Therefore, there is an urgent need to provide an assay for the detection of impurities in vorexant fumarate. However, pyridine-3-sulfonic acid has a boiling point above 330 ℃ which exceeds the temperature range of most gas chromatography columns and is therefore unsuitable for measurement by gas Chromatography (CG); and the sulfonic acid ester has high reactivity, and the sulfonic acid ester can be decomposed at high temperature at the injection port or the sulfonic acid in the sample can be instantaneously reacted with the alcohol solvent to generate the sulfonic acid ester when GC is used for detection. When the HPLC method is adopted for determination, the residual sulfonic acid in the sample is easy to react with an alcohol solvent commonly used in the test to generate sulfonate to be determined, so that a false positive result is caused, and the accuracy of the determination result is influenced; and the sulfonate impurities have high polarity and are weakly retained in a chromatographic column, and can be easily eluted, so that the impurities cannot be effectively separated. For example, a method for measuring the content of pyridine-3-sulfonic acid in a bulk drug of vonoprazan fumarate by using an HPLC method (Lezhiyan, Xiaomin, Chuanjiejing. HPLC method is used for measuring the content of pyridine-3-sulfonic acid [ J ] in the bulk drug of vonoprazan fumarate, and 2019, v.29(01):23-26.) is disclosed, under a selected chromatographic condition, the pyridine-3-sulfonic acid is well separated from adjacent chromatographic peaks, the peak area shows a good linear relation, the precision is good, the sensitivity is high, and the accuracy is good, but the method can only detect the pyridine-3-sulfonic acid, the pyridine-3-methyl sulfonate and the pyridine-3-ethyl sulfonate have no response values and cannot detect simultaneously.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the prior art that CG or HPLC (high performance liquid chromatography) cannot be used for simultaneously determining 3 impurities of pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate in Voranol fumarate, and provides a detection method for simultaneously determining 3 impurities of pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate in Voranol fumarate.
The invention aims to provide a detection method for simultaneously determining 3 impurities of pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate in Voranol fumarate.
The above purpose of the invention is realized by the following technical scheme:
a method for simultaneously detecting 3 impurities of pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate in Voranolan fumarate adopts high performance liquid chromatography for detection,
wherein the structures of the pyridine-3-sulfonic acid, the pyridine-3-sulfonic acid methyl ester and the pyridine-3-sulfonic acid ethyl ester are as follows:
wherein, the conditions of the high performance liquid chromatography are as follows:
a chromatographic column filled with octadecylsilane chemically bonded silica is adopted, under the conditions of a detection wavelength of 260nm, a column temperature of 25-45 ℃ and a flow rate of 0.8-1.2 ml/min, a dipotassium hydrogen phosphate solution of 0.02-0.05 mol/L is used as a mobile phase A, acetonitrile is used as a mobile phase B, and detection is carried out under the condition of gradient elution.
Further, the gradient elution is 0-4 min, and the mobile phase A: 98-98%; 4-10 min, mobile phase A: 98-40%; 10-20 min, mobile phase A: 40-40%; 20-20.1 min, mobile phase A: 40-98%; 20.1-25 min, mobile phase A: 98-98%.
Preferably, the reverse phase C18 chromatographic column is Sepax HP-C184.6mm X250 mm, 5 μm.
Preferably, when the Voranolan fumarate is measured, the test solution is prepared by using a 2-5% acetonitrile solution.
More preferably, the test solution is prepared with a 5% acetonitrile solution in the assay of vorexant fumarate.
Further, the concentration of the Voranolan fumarate in the test solution is 0.5-4 mg/ml.
Preferably, the pH value of the dipotassium hydrogen phosphate solution is adjusted to 3.0-6.0 by using phosphoric acid.
More preferably, the dipotassium hydrogen phosphate solution is adjusted to a pH of 6.0 with phosphoric acid.
Preferably, the column temperature is 25-30 ℃; more preferably, the column temperature is 30 ℃.
Preferably, the flow rate is 0.8-1.0 ml/min; more preferably, the flow rate is 1.0 ml/min.
Preferably, the mobile phase A is 0.05mol/L dipotassium hydrogen phosphate solution.
Furthermore, the sample injection amount is 50-100 μm under the high performance liquid chromatography condition.
Furthermore, in the detection method, the limit of quantification of pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate is 0.001-0.002%, and the limit of detection is 0.0004-0.0007%; the limit of the quantification of the pyridine-3-sulfonic acid is 0.08-0.10%, and the limit of the detection is 0.004-0.005%.
Furthermore, in the detection method, the preparation method of the test solution comprises the following steps: taking a proper amount of the voronol fumarate, dissolving the appropriate amount of the voronol fumarate in a 2-5% acetonitrile solution, and diluting to prepare a solution containing 0.5-4 mg of the voronol fumarate per 1ml, wherein the solution is used as a test solution.
Further, in the detection method, the preparation method of the reference solution comprises the following steps: taking a proper amount of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid reference substances, and preparing a solution containing 0.014-0.112 mu g of pyridine-3-methyl sulfonate, 0.014-0.112 mu g of pyridine-3-ethyl sulfonate and 0.375-3 mu g of pyridine-3-sulfonic acid in each 1ml of acetonitrile solution with the concentration of 2-5%.
The invention has the following beneficial effects:
the detection method can simultaneously detect 3 impurities of pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate in Voranolan fumarate under the condition of one chromatogram, has better separation degree of each chromatogram peak, high sensitivity, higher recovery rate, precision and stability, good reproducibility and high accuracy, meets the qualitative and quantitative analysis requirements of the medicine, can be used for monitoring the impurities in the production process of Voranolan fumarate, and is beneficial to controlling and improving the quality of Voranolan fumarate; meanwhile, the method is simple to operate, has a detection time period, and is very suitable for quality monitoring of the bulk drugs in large-scale industrial production.
Drawings
FIG. 1 is a chromatogram for detecting a labeled test solution in example 1 of the present invention.
FIG. 2 is a partially enlarged view of a detection chromatogram of the labeled test solution in example 1 of the present invention.
FIG. 3 is a chromatogram for detecting the solution of the standard test sample in comparative example 1.
FIG. 4 is a cross-sectional view of the pyridine-3-sulfonic acid chromatogram peak in the detection chromatogram of the control solution in comparative example 3 of the present invention.
FIG. 5 is a chromatogram of a different mobile phase in comparative example 4 according to the invention.
FIG. 6 is a chromatogram of the measurement of a sample solution randomly sampled and inspected in application example 2 of the present invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 detection method for simultaneously determining 3 impurities pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate in Voranolan fumarate
(1) Conditions of liquid chromatography
Mobile phase A: 0.05mol/L dipotassium hydrogen phosphate solution (pH adjusted to 6.0 with phosphoric acid)
Mobile phase B: acetonitrile
See table 1 for elution gradients.
TABLE 1 gradient elution conditions
The detection wavelength is 260nm, the column temperature is 30 ℃, the flow rate is 1.0ml/min, and the sample injection amount is 50 mul;
a chromatographic column: sepax HP-C184.6mm. times.250 mm, 5 μm.
(2) Preparation of test and reference substances
Test solution: a proper amount of the vorexant fumarate is dissolved and diluted by a 5% acetonitrile solution to prepare a solution containing about 2mg of the vorexant fumarate per 1ml, and the solution is used as a test solution.
Adding a standard test solution: taking a proper amount of reference substances of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid, preparing a solution containing 1.5 mu g, 1.5 mu g and 15 mu g of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid in each 1ml of acetonitrile solution with 5 percent as a reference substance stock solution; taking a proper amount of the vorexant fumarate, adding a proper amount of 5% acetonitrile solution to dissolve the vorexant fumarate, precisely adding a proper amount of a reference substance stock solution, diluting the reference substance stock solution with the 5% acetonitrile solution to prepare solutions containing 0.056 mu g of pyridine-3-methyl sulfonate, 0.056 mu g of pyridine-3-ethyl sulfonate and 2mg of pyridine-3-sulfonic acid in each 1ml of the solution, and taking the solutions as a sample solution.
Preparation of a reference solution: taking a proper amount of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid reference substances, and preparing a solution containing 0.056 mu g, 0.056 mu g and 1.5 mu g of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid in each 1ml of acetonitrile solution with 5 percent as a reference solution.
(3) Adding standard sample solution and reference substance for detection and result
Taking a 5% acetonitrile solution, a standard sample solution and a reference substance solution, and carrying out sample injection detection under the liquid chromatography condition (1), wherein the detection results are shown in tables 2-3 and figure 1.
TABLE 2 result table of separation degree of sample solution with added standard
| Name (R) | Retention time/min | Degree of separation |
| Fumaric acid | 2.913 | - |
| Pyridine-3-sulfonic acid methyl ester | 3.441 | 3.12 |
| Pyridine-3-sulfonic acid ethyl ester | 4.083 | 3.85 |
| Pyridine-3-sulfonic acid | 5.562 | 5.29 |
TABLE 3 results of control solutions
| Name (R) | Retention time/min | Degree of separation |
| Pyridine-3-sulfonic acid methyl ester | 3.305 | - |
| Pyridine-3-sulfonic acid ethyl ester | 3.960 | 4.81 |
| Pyridine-3-sulfonic acid | 5.930 | 5.68 |
The results show that the blank solvent (5% acetonitrile solution) does not interfere with the determination of pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester and pyridine-3-sulfonic acid; in the solutions of the standard sample and the reference substance, the separation degrees of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid and adjacent peaks meet the requirements.
(4) Detection Limit and detection Limit determination
And (3) taking the reference substance solution in the step (2) for dilution step by step, and inspecting the detection Limit (LOD) and the quantification Limit (LOQ) of the impurities, wherein the results are shown in tables 4-5.
TABLE 4 quantitative limit test results
| Impurities | Peak area | S/N | Quantitative limit/%) |
| Pyridine-3-sulfonic acid methyl ester | 956 | 15 | 0.001 |
| Pyridine-3-sulfonic acid ethyl ester | 1518 | 22 | 0.001 |
| Pyridine-3-sulfonic acid | 6944 | 42 | 0.010 |
TABLE 5 detection Limit results
| Impurities | Peak area | S/N | Detection limit/%) |
| Pyridine-3-sulfonic acid methyl ester | 448 | 5 | 0.0004 |
| Pyridine-3-sulfonic acid ethyl ester | 835 | 10 | 0.0004 |
| Pyridine-3-sulfonic acid | 3129 | 15 | 0.0051 |
As can be seen from the table, the detection method has lower detection limit and quantification limit for pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid, and has high detection sensitivity.
Example 2 detection method for simultaneously determining 3 impurities pyridine-3-sulfonic acid, pyridine-3-sulfonic acid methyl ester and pyridine-3-sulfonic acid ethyl ester in Voranol fumarate
Example 2 the detection method differs from that of example 1 in that the mobile phase a: a0.05 mol/L dipotassium hydrogenphosphate solution (pH adjusted to 6.0 with phosphoric acid) was replaced with a 0.02mol/L dipotassium hydrogenphosphate solution (pH adjusted to 6.0 with phosphoric acid), and the rest of the parameters were referred to in example 1. The results of the resolution of the spiked test solutions are shown in Table 6.
TABLE 6 results of degree of separation of solutions of spiked test samples
| Name (R) | Retention time/min | Degree of separation |
| Fumaric acid | 2.951 | - |
| Pyridine-3-sulfonic acid methyl ester | 3.436 | 3.03 |
| Pyridine-3-sulfonic acid ethyl ester | 4.076 | 3.78 |
| Pyridine-3-sulfonic acid | 5.536 | 5.29 |
As can be seen from the table, the degrees of separation of pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester and pyridine-3-sulfonic acid from adjacent peaks are satisfactory.
Example 3 detection method for simultaneously determining 3 impurities pyridine-3-sulfonic acid, pyridine-3-sulfonic acid methyl ester and pyridine-3-sulfonic acid ethyl ester in Voranol fumarate
Example 3 the detection method differs from example 1 in that the mobile phase a: a0.05 mol/L potassium hydrogenphosphate solution (pH adjusted to 6.0 with phosphoric acid) was replaced with a 0.05mol/L potassium hydrogenphosphate solution (pH adjusted to 3.0 with phosphoric acid), and the rest of the parameters were referred to in example 1. The results of the resolution of the spiked test solutions are shown in Table 7.
TABLE 7 results of degree of separation of solutions of spiked test samples
| Name (R) | Retention time/min | Degree of separation |
| Fumaric acid | 3.058 | - |
| Pyridine-3-sulfonic acid methyl ester | 3.438 | 3.10 |
| Pyridine-3-sulfonic acid ethyl ester | 4.081 | 3.86 |
| Pyridine-3-sulfonic acid | 5.548 | 5.27 |
As can be seen from the table, the degrees of separation of pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester and pyridine-3-sulfonic acid from adjacent peaks are satisfactory.
Example 4 detection method for simultaneous determination of 3 impurities pyridine-3-sulfonic acid, pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester in Voranolan fumarate
Example 4 the difference between the detection method and example 1 is that the flow rate in the liquid chromatography conditions of (1) in example 1 was changed to 0.8ml/min, the column temperature was changed to 45 deg.c, and the remaining parameters were referred to in example 1. The results of the resolution of the spiked test solutions are shown in Table 8.
TABLE 8 results of degree of separation of solutions of spiked test samples
As can be seen from the table, the degrees of separation of pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester and pyridine-3-sulfonic acid from adjacent peaks are satisfactory.
Example 5A detection method for simultaneously determining 3 impurities pyridine-3-sulfonic acid, pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester in Voranolan fumarate
Example 5 the difference between the detection method and example 1 is that the flow rate in the liquid chromatography conditions of (1) in example 1 was changed to 1.2ml/min, the column temperature was changed to 25 ℃, and the rest of the parameters were referred to in example 1. The results of the resolution of the spiked test solutions are shown in Table 9.
TABLE 9 results of degree of separation of solutions of spiked test samples
| Name (R) | Retention time/min | Degree of separation |
| Fumaric acid | 2.774 | - |
| Pyridine-3-sulfonic acid methyl ester | 3.036 | 2.76 |
| Pyridine-3-sulfonic acid ethyl ester | 3.809 | 3.53 |
| Pyridine-3-sulfonic acid | 5.593 | 5.84 |
As can be seen from the table, the degrees of separation of pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester and pyridine-3-sulfonic acid from adjacent peaks are satisfactory.
Example 6A detection method for simultaneously determining 3 impurities pyridine-3-sulfonic acid, pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester in Voranolan fumarate
Example 6 the detection method is different from example 1 in that the concentration of the solution of the control in example 1(2) is adjusted to 0.5mg/ml according to the concentration of the test sample, and the rest parameters refer to example 1. The results of the quantitation limit and detection limit are shown in tables 10-11.
TABLE 10 detection of quantitative limit
| Impurities | Peak area | S/N | Quantitative limit/%) |
| Pyridine-3-sulfonic acid methyl ester | 357 | 12 | 0.002 |
| Pyridine-3-sulfonic acid ethyl ester | 436 | 15 | 0.002 |
| Pyridine-3-sulfonic acid | 1309 | 21 | 0.008 |
TABLE 11 detection limit test results
| Impurities | Peak area | S/N | Detection limit/%) |
| Pyridine-3-sulfonic acid methyl ester | 184 | 3 | 0.0008 |
| Pyridine-3-sulfonic acid ethyl ester | 176 | 4 | 0.0007 |
| Pyridine-3-sulfonic acid | 651 | 7 | 0.004 |
As can be seen from the table, the detection method has lower detection limit and quantification limit for pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid, and has high detection sensitivity.
Example 7 detection method for simultaneous determination of 3 impurities pyridine-3-sulfonic acid, pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester in Voranolan fumarate
Example 7 the detection method differs from example 1 in that the 5% acetonitrile solution flowing in example 1(2) was replaced with a 2% acetonitrile solution, and the remaining parameters refer to example 1. The results of the control solution tests are shown in Table 12.
TABLE 12 control solution separation results
| Name (R) | Retention time/min | Peak area |
| Pyridine-3-sulfonic acid methyl ester | 3.221 | 3440 |
| Pyridine-3-sulfonic acid ethyl ester | 3.883 | 3789 |
| Pyridine-3-sulfonic acid | 5.648 | 68560 |
The result shows that the blank solvent (2% acetonitrile solution) does not interfere the determination of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid, and pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid have stronger response values.
Comparative example 1 detection method for determining impurities in Vonopalatine fumarate
Comparative example 1 the detection method is different from that of example 1 in that the column in the (1) liquid chromatography condition of example 1 is changed to Waters Symmetry C18, 4.6mm X250 mm, 5 μm, and the rest of the parameters refer to example 1. The results of the resolution of the spiked test solutions are shown in Table 13 and FIG. 2.
TABLE 13 results of degree of separation of solutions of spiked test samples
| Name (R) | Retention time/min | Degree of separation |
| Fumaric acid | 2.252 | - |
| Pyridine-3-sulfonic acid methyl ester | 2.507 | No baseline separation |
| Pyridine-3-sulfonic acid ethyl ester | 2.943 | - |
| Pyridine-3-sulfonic acid | 4.636 | 10.45 |
As can be seen from the table and the figure, the peak of the pyridine-3-sulfonic acid methyl ester is not completely separated from the peak of the fumaric acid, the separation degree is not satisfactory, and the separation degree cannot be calculated from the pyridine-3-sulfonic acid ethyl ester.
Comparative example 2 detection method for determining impurities in Vonopalatine fumarate
Comparative example 2 the detection method is different from that of example 1 in that the 5% acetonitrile solution of the stream in example 1(2) is replaced with 10%, 20%, 25% acetonitrile solution, respectively, and the rest of the parameters refer to example 1.
The results show that after the 5% acetonitrile solution is replaced by the 10%, 20% and 25% acetonitrile solution, respectively, the pyridine-3-sulfonic acid ethyl ester has the effect of 3.840min, and has interference peaks with peak areas of 300, 786 and 852.
Comparative example 3 detection method for determining impurities in Vonopalatine fumarate
Comparative example 3 the test method was different from that of example 1 in that the 5% acetonitrile solution of the stream in example 1(2) was replaced with 50% methanol solution and 50% acetonitrile solution, respectively, and 5% acetonitrile solution was used as a comparison, and the rest of the parameters were as in example 1. See table 14 and fig. 3 for the control solution test results.
TABLE 14 test results of control solutions
| Sample preparation solvent | Peak area for pyridine-3-sulfonic acid detection |
| 50% acetonitrile | 2007 |
| 50% methanol | 3548 |
| Sample preparation solvent of example 1 5% acetonitrile | 72894 |
As can be seen from the table and the figure, when the control solution is prepared by using a 50% methanol solution and a 50% acetonitrile solution, the response value of the detection peak area of pyridine-3-sulfonic acid is very low, and the detection requirement is not met.
Comparative example 4 detection method for determining impurities in Vonopalatine fumarate
Comparative example 4 the detection method differs from that of example 1 in that the mobile phase in the liquid chromatography conditions of (1) of example 1 is changed to:
mobile phase A: 0.022mol/L sodium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (90: 10)
Mobile phase B: 0.022mol/L sodium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (30: 70)
The rest parameters are referred to example 1, wherein pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester and pyridine-3-sulfonic acid in the reference solution are respectively prepared and detected according to the concentration, and the detection results are shown in fig. 5.
As can be seen from the figure, pyridine-3-sulfonic acid methyl ester and pyridine-3-sulfonic acid ethyl ester in the detection method all peak at 1.9min (in the dead volume of the instrument), and the initial proportion of the method is too high compared with the organic phase, which is not suitable.
Comparative example 5 detection method for determining impurities in Vonopalatine fumarate
Comparative example 5 the detection method differs from that of example 1 in that the mobile phase a in the liquid chromatography conditions of (1) of example 1 is changed to: 0.1% formic acid solution, the rest parameters refer to example 1, and the results of the test of the control solution are shown in Table 15.
TABLE 15 test results of control solutions
| Name (R) | Retention time/min |
| Pyridine-3-sulfonic acid methyl ester | 0.907 |
| Pyridine-3-sulfonic acid ethyl ester | 0.977 |
| Pyridine-3-sulfonic acid | 1.120 |
As can be seen from the table, pyridine-3-sulfonic acid methyl ester and pyridine-3-sulfonic acid ethyl ester in the detection method all peak at 1min, and cannot be separated.
Application example 1 methodological investigation
(1) Linear and Range testing
Respectively and precisely measuring appropriate amounts of reference substance stock solutions in example 1(2) to respectively prepare reference substance solutions with the concentrations of pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate of 0.01-0.1 mug/ml and pyridine-3-sulfonic acid of 0.3-3 mug/ml; the liquid chromatography conditions of example 1(1) were followed and the sample injection was examined. And (3) performing linear regression treatment by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and calculating a regression equation and a correlation coefficient, wherein the results are shown in tables 16-18.
TABLE 16 pyridine-3-sulfonic acid methyl ester linearity results
TABLE 17 linearity of pyridine-3-sulfonic acid ethyl ester results
TABLE 18 pyridine-3-sulfonic acid linearity results
As can be seen from the table, the pyridine-3-methyl sulfonate is between 0.0115 mug/ml and 0.1145 mug/ml, the peak area and the concentration have good linear relation, wherein r is 0.9998, and the ratio of the response value of the Y-axis intercept at 100% concentration is 2.9%; the pyridine-3-ethyl sulfonate is between 0.0117 mu g/ml and 0.1167 mu g/ml, the peak area and the concentration have a good linear relation, wherein r is 0.9984, and the ratio of the Y-axis intercept at 100% concentration response value is 0.9%; the pyridine-3-sulfonic acid is between 0.3067 mu g/ml and 3.0668 mu g/ml, the peak area and the concentration have good linear relation, wherein r is 1.0000, and the ratio of the response value of the Y-axis intercept at 100 percent is 0.3 percent.
(2) Precision test
The control solution was continuously injected 6 times under the conditions of example 1, and the peak areas and RSD of pyridine-3-sulfonic acid methyl ester, pyridine-3-sulfonic acid ethyl ester and pyridine-3-sulfonic acid were calculated, and the results are shown in Table 19.
TABLE 19 sample introduction precision results for control
As can be seen from the table, the reference substance solution detected by the detection method of the invention has the main peak area RSD less than 10.0% and good precision.
(3) Accuracy test
According to the preparation method of the sample solution added with the standard in the step (2) of example 1, 6 parts of sample solution added with the standard with limited concentration (the concentration of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid is 0.056. mu.g/ml, 0.056. mu.g/ml and 1.5. mu.g/ml respectively) are prepared, and the sample injection detection is carried out according to the liquid chromatography condition in the step (1) of example 1. The contents of pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate are calculated by peak area according to an external standard method, and the results are shown in tables 20-21.
TABLE 20 results of recovery of methyl and ethyl pyridine-3-sulfonate
TABLE 21 pyridine-3-sulfonic acid recovery results
As can be seen from the table, the average recovery rate of pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate is 83%, the single recovery rate is between 75% and 90%, and the RSD is 6.4%; the average recovery rate of the pyridine-3-sulfonic acid is 100 percent, the single recovery rate is between 99 percent and 101 percent, and the RSD is 0.4 percent; are all less than 20.0 percent, and have high accuracy.
(4) Stability test of solution
Control solutions were prepared according to the preparation method in step (2) of example 1, and the control solutions were allowed to stand at room temperature for 0h, 4h, 8h, and 12h, respectively, and then injected into a liquid chromatograph, a chromatogram was recorded, and the relative change rate of the peak area of each impurity at each time point as compared with the peak area at 0h was measured and calculated, and the results are shown in table 22.
TABLE 22 control solution stability results
As can be seen from the table, compared with 0h, after the control solution is placed for 8 hours, the peak area change rates of pyridine-3-methyl sulfonate, pyridine-3-ethyl sulfonate and pyridine-3-sulfonic acid are all less than 5%, and the control solution is stable when placed for 8 hours at room temperature.
Application example 2 test article solution measurement
Application example 2 the test methods and conditions of example 1 were used to test a random sample test solution, and the results are shown in fig. 6.
As can be seen, pyridine-3-sulfonic acid ethyl ester and pyridine-3-sulfonic acid impurities exist in the test sample and are detected by the method; wherein, the content of pyridine-3-sulfonic acid ethyl ester is lower than that of pyridine-3-sulfonic acid, but the method still has response values which can be detected and has better separation degree.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. A detection method for simultaneously determining 3 impurities in Vonopalatine fumarate is characterized in that high performance liquid chromatography is adopted for detection, wherein the 3 impurities are pyridine-3-sulfonic acid, pyridine-3-methyl sulfonate and pyridine-3-ethyl sulfonate;
the high performance liquid chromatography conditions are as follows:
adopting a reverse phase C18 chromatographic column Sepax HP-C184.6 mm multiplied by 250mm, 5 mu m, under the conditions of 260nm detection wavelength, 25-45 ℃ column temperature and 0.8-1.2 ml/min flow rate, taking 0.02-0.05 mol/L dipotassium hydrogen phosphate as a mobile phase A and acetonitrile as a mobile phase B, and carrying out detection under the condition of gradient elution;
adjusting the pH value of the dipotassium phosphate solution to 3.0-6.0 by using phosphoric acid;
the gradient elution is 0-4 min, and the mobile phase A: 98-98%; 4-10 min, mobile phase A: 98-40%; 10-20 min, mobile phase A: 40-40%; 20-20.1 min, mobile phase A: 40-98%; 20.1-25 min, mobile phase A: 98-98%;
when the Voranolan fumarate is measured, a test solution is prepared by using a 2-5% acetonitrile solution.
2. The assay method according to claim 1, wherein the test solution is prepared from a 5% acetonitrile solution in the assay of Voranolan fumarate.
3. The detection method according to claim 1, wherein the concentration of vorexan fumarate in the sample solution is 0.5 to 4 mg/ml.
4. The detection method according to claim 1, wherein the dipotassium phosphate solution is adjusted to a pH of 6.0 with phosphoric acid.
5. The detection method according to claim 1, wherein the amount of sample is 50 to 100 μm under the high performance liquid chromatography condition.
6. The detection method according to claim 1, wherein the limit of quantitation of pyridine-3-sulfonic acid methyl ester and pyridine-3-sulfonic acid ethyl ester in the detection method is 0.001-0.002%, and the limit of detection is 0.0004-0.0007%; the limit of the quantification of the pyridine-3-sulfonic acid is 0.008-0.010%, and the limit of the detection is 0.004-0.005%.
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