CN111595996A - Detection method and application of camostat mesylate related substance - Google Patents
Detection method and application of camostat mesylate related substance Download PDFInfo
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Abstract
The invention provides a detection method and application of related substances of camostat mesylate, wherein the method is liquid chromatography, and a chromatographic column is an octadecylsilane chemically bonded silica chromatographic column; the mobile phase A is ion pair solution, and the mobile phase B is one or two of acetonitrile, methanol and ethanol; the detector is a diode array detector, and samples are fed and analyzed according to a certain amount of sample concentration. The method is applied to the detection of related substances of a starting material in a camostat mesylate synthesis process, the detection of the residual quantity of a catalyst 4-dimethylaminopyridine used in the synthesis process and the detection of the residual quantity of refined solvents acetone and DMF.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly relates to a liquid phase detection method and application of related substances of camostat mesylate.
Background
The chemical name of the methanesulfonic acid Camostat (English name: Camostat Meilate) is 2- (dimethylamino) -2-oxoethyl 4- (4-guanidinobenzoyloxy) phenylacetate methanesulfonic acid salt, and the molecular formula is C20H22N4O5CH4O3S, molecular weight 494.52, CAS registry number 59721-29-8. The structure is shown as formula I:
camostat mesylate is a non-peptide protease inhibitor developed by nippon drug corporation, and its action mechanism is that after oral administration, camostat mesylate quickly acts on the kinin production system, fibrinolysis system, coagulation system and complement system of the body, and inhibits the hyperfunction of the enzyme activity of these systems, thereby controlling the symptoms of chronic pancreatitis, relieving pain and reducing amylase value. In addition, the product can be used for treating diffuse blood vessel coagulation, and also has anticancer and urine protein reducing effects. Further research indicates that the camostat mesylate as a serine protease inhibitor shows an inhibition effect in cell experiments, can block new coronavirus from infecting lung cells, and is expected to become a potential small molecule drug.
The camostat mesylate takes p-aminobenzoic acid as an initial material and reacts with cyanamide under certain conditions to prepare p-guanidinobenzoic acid hydrochloride. N, N-dimethyl chloroacetamide is taken as a raw material, p-hydroxyphenylacetic acid-N, N-dimethyl carbamyl methyl ester is prepared by condensation with p-hydroxyphenylacetic acid, p-guanidinobenzoic acid hydrochloride and p-hydroxyphenylacetic acid-N, N-dimethyl carbamyl methyl ester are condensed under certain conditions to obtain camostat mesylate hydrochloride, sodium bicarbonate is used for treating camostat carbonate, and the camostat carbonate is acidified by methanesulfonic acid under certain conditions to prepare camostat mesylate (the process is introduced in the synthesis process of camostat mesylate, a drug for treating pancreatitis, published in chemical engineering by Chenbao quan and the like).
Aminobenzoic acid, N, N-dimethyl chloroacetamide and p-hydroxyphenylacetic acid are starting materials for synthesizing camostat mesylate, p-aminobenzoic acid reacts with cyanamide to obtain p-guanidinobenzoic acid hydrochloride, and the p-guanidinobenzoic acid hydrochloride further reacts with the p-hydroxyphenylacetic acid, so the p-aminobenzoic acid and the cyanamide can be regarded as the starting materials for synthesizing the p-guanidinobenzoic acid hydrochloride, the camostat mesylate does not need to carry out quality control on the aminobenzoic acid and the cyanamide, and the N, N-dimethyl chloroacetamide does not have an ultraviolet absorption functional group and can be controlled in residual solvent detection. P-guanidinobenzoic acid hydrochloride and p-hydroxyphenylacetic acid can therefore be considered as starting materials for the synthesis of camostat mesylate. In addition, camostat mesylate has poor stability and is unstable in acid, alkali, oxidation, illumination and heating environments, and can be degraded into guanidinobenzoic acid hydrochloride, p-hydroxyphenylacetic acid-N, N-dimethylcarbamoylmethyl ester, degradation impurity A and degradation impurity B, and the degradation impurity A and the degradation impurity B are also process impurities in the camostat mesylate synthesis process. Therefore, a method for simultaneously detecting the contents of guanidinobenzoic acid hydrochloride, p-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester, degradation impurity A and degradation impurity B in the camostat mesylate is needed to be established, and the quality of related substances of the camostat mesylate is controlled. The camostat mesylate has the potential of multiple indications and is a small-molecule drug for potential treatment of new coronary pneumonia, and a method for establishing quality control of the camostat mesylate related substances is also very necessary.
The degradation impurity A is 4- (4-guanidinobenzoyloxy) phenylacetic acid mesylate, and the structural formula is as follows:
the degradation impurity B is 4- (4-guanidinobenzoyloxy) phenylacetic acid [2- (dimethylamino) -2-oxoethyl ] ester trifluoroacetate, and the structural formula of the degradation impurity B is as follows:
disclosure of Invention
The invention aims to provide a liquid phase method for detecting related substances of camostat mesylate and application of the method in detection of related substances of starting materials in a camostat mesylate synthesis process, residual quantity of a catalyst 4-dimethylaminopyridine used in the synthesis process and residual quantity of refined solvents acetone and DMF.
In order to achieve the purpose, the invention discloses the following technical contents:
a method for detecting related substances of camostat mesylate is characterized by comprising the following steps: the detection method is liquid chromatography, and the chromatographic conditions are as follows:
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column;
the mobile phase A is: the ion pair solution, the mobile phase B is one or two of acetonitrile, methanol and ethanol;
the diluent is: pure water or salt solution, or a mixed solution of pure water or salt solution and one or two of methanol, acetonitrile and ethanol;
the ion pair solution refers to: adding a certain amount of acid into a mixed solution of sodium heptanesulfonate, sodium dodecyl sulfate and tetrabutylammonium hydrogen sulfate;
the acid refers to: any one or more of acetic acid, phosphoric acid, methanesulfonic acid, boric acid, and formic acid;
the salt solution is one or a mixture of two of disodium hydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and ammonium formate;
the detector is one of an ultraviolet detector and a diode array detector; the detection wavelength of the detector is 210 nm-280 nm;
the chromatographic column is selected from: kromasil C18, ODS-C18, Thermo Syncronis C18, Thermo Hypersil GOLD C18, Waters symmetry C18, Waters RP C18, Agilent XDB C18, Agilent eclipse plus C18, Shimadzu GL ODS C18, Waters BEH C18; the flow rate of the mobile phase is 0.8-1.2 ml/min; the column temperature is 30-40 ℃; the preferred column is Waters BEH C18. According to the invention, a certain amount of sample concentration is used for sample injection analysis, and the sample concentration is 0.5-3.0 mg/ml;
the mobile phase A: and carrying out gradient elution on the mobile phase B, wherein the elution ratio is as follows:
the ion pair solution of the preferred mobile phase A contains 0.5-1.5% of sodium heptanesulfonate, 0.05-0.5% of sodium dodecyl sulfate and 1-4% of tetrabutylammonium hydrogen sulfate; the percentage is mass percentage, and 0.05% -1.0% acetic acid, the percentage is volume percentage; the mobile phase B is methanol. More preferably, the ion pair solution of mobile phase a contains 0.5% sodium heptanesulfonate, 0.1% sodium lauryl sulfate, 2% tetrabutylammonium hydrogen sulfate and 0.1% acetic acid.
The diluent is a mixed solution of 0.005-0.1 mol/L dipotassium hydrogen phosphate solution and methanol, wherein the pH value of the mixed solution is 5.0-7.5; the content of the methanol in the mixed solution is 10% -60%, and the percentage is volume percentage. The pH value of the potassium dihydrogen phosphate solution is 7.0, and the concentration is 0.01 mol/L. The detector is a diode array detector; the detection wavelengths are as follows:
the invention further discloses an application of the detection method of the related substances of the camostat mesylate in the detection of the related substances of the starting materials in the camostat mesylate synthesis process. In particular to the application of the method in the detection of the residual quantity of 4-dimethylamino pyridine used as a catalyst for a synthesis process and the residual quantity of acetone and DMF used as refined solvents. The experimental results show that: has good specificity, sensitivity and accuracy when controlling related substances of the starting materials and controlling the residual quantity of acetone and DMF.
The invention is described in more detail below:
the chromatographic conditions were as follows:
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column selected from: kromasil C18, ODS-C18, ThermoSyncronis C18, Thermo Hypersil GOLD C18, Waters symmetry C18, Waters RP C18, Agilent XDB C18, Agilent Eclipse Plus C18, Shimadzu GL ODS C18.
The mobile phase A is ion pair solution, and the mobile phase B is one or two of acetonitrile, methanol and ethanol;
the detector is one of an ultraviolet detector and a diode array detector;
preferably, the sample concentration is 0.5-3.0 mg/ml;
preferably, the diluent is a mixed solution of a salt solution and methanol;
further, the mixed solution of the salt solution and the methanol contains 10% -60% of methanol. The percentage is volume percentage;
furthermore, the salt solution contains dipotassium hydrogen phosphate with the salt concentration of 0.01mol/L, and the pH value of the salt solution is 7.0;
preferably, the chromatography column is Waters BEH C18;
further, the flow rate of the chromatographic column is 0.8-1.2ml/min, and the column temperature is 30-40 ℃.
Preferably, the mobile phase A is an ion-pair solution containing 0.5% of sodium heptanesulfonate, 0.1% of sodium dodecyl sulfate and 2% of tetrabutylammonium hydrogen sulfate, and an amount of acetic acid is added, wherein the percentage is the mass fraction. The mobile phase B is methanol.
Further, the amount of acetic acid added is 0.1% acetic acid, and the percentages are volume fractions.
Further, mobile phase a and mobile phase B were subjected to gradient elution as shown in table 1:
TABLE 1 gradient elution
Preferably, the detector is a diode array detector.
Preferably, the detection wavelength of the detector is as shown in table 2:
TABLE 2 detection wavelength
The detection method of the related substances of the camostat mesylate provided by the invention adopts a high performance liquid chromatography to realize the perfect separation of p-guanidinobenzoic acid, p-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester, process impurities and degradation impurities, has higher sensitivity and specificity, is simple and convenient to operate, and has the minimum separation degree of more than 2.0. Therefore, the method can be used for quality control of related substances of camostat mesylate, and can detect the residual quantity of the catalyst 4-dimethylaminopyridine used in the synthesis process and the refined solvents acetone and DMF. Has practical significance.
The invention mainly solves the problem that the detection method of related substances of the camostat mesylate is difficult to develop, and the Japanese pharmacopoeia only adopts simple thin-layer chromatography for control. The specificity, accuracy, precision and durability of the detection method are mainly considered. The main difficulty lies in thoroughly solving the problems that the retention time of impurities is too short or too long due to the fact that related substances of the camostat mesylate have large polarity difference and are not easy to retain or too long to retain, and the specificity, accuracy, precision and durability of the method are influenced.
Drawings
FIG. 1 is a liquid chromatogram of a diluent-positioning solution;
FIG. 2 is a liquid chromatogram of p-hydroxyphenylacetic acid positioning solution;
FIG. 3 is a liquid chromatogram of a positioning solution of p-guanidinobenzoic acid;
FIG. 4 is a liquid chromatogram of p-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester positioning solution;
FIG. 5 is a liquid chromatogram of an impurity A localization solution;
FIG. 6 is a liquid chromatogram of an impurity B localization solution;
FIG. 7 is a liquid chromatogram of a camostat mesylate positioning solution;
FIG. 8 is a liquid chromatogram of the mixed solution;
FIG. 9 is a graph of p-hydroxyphenylacetic acid linearity and range;
FIG. 10 is a graph of the linearity and range of p-guanidinobenzoic acid;
FIG. 11 is a graph of the linearity and range of p-hydroxyphenylacetic acid-N, N-dimethylcarbamoylmethyl ester;
FIG. 12 is a graph of the linearity and range of degradation of impurity A;
FIG. 13 is a graph of the linearity and range of degraded impurity B;
figure 14 is a plot of camostat mesylate linearity and range.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.
Example 1
The experimental conditions are as follows:
waters ultra high performance liquid chromatograph;
a chromatographic column: waters BEH C182.1X 100mm 1.7 μm;
flow rate: 0.2 mL/min;
column temperature: 30 ℃;
sample introduction amount: 2 mu L of the solution;
sample concentration: 3 mg/ml;
diluent agent: 0.01mol/L dipotassium hydrogen phosphate (pH = 7.0) solution: methanol =1: 1;
detection wavelength:
mobile phase A: 0.75% of sodium heptanesulfonate, 0.08% of sodium dodecyl sulfate, 1.5% of tetrabutylammonium hydrogen sulfate (mass fraction) and 0.15% of acetic acid (volume fraction).
Mobile phase B: methanol
TABLE 3 results of the experiment
As can be seen from table 3, the separation of each compound was good.
Example 2
The experimental conditions are as follows:
waters ultra high performance liquid chromatograph;
a chromatographic column: waters BEH C182.1X 100mm 1.7 μm;
flow rate: 0.2 mL/min;
column temperature: 35 ℃;
sample introduction amount: 2 mu L of the solution;
sample concentration: 3 mg/ml;
diluent agent: 0.01mol/L dipotassium hydrogen phosphate (pH = 7.0) solution: methanol =1: 1;
detection wavelength:
mobile phase A: 0.05% of sodium heptanesulfonate, 0.10% of sodium dodecyl sulfate, 2.5% of tetrabutylammonium hydrogen sulfate (mass fraction) and 0.10% of acetic acid (volume fraction).
Mobile phase B: methanol
TABLE 4 results of the experiment
As can be seen from table 4, the separation of each compound was good.
Example 3
The experimental conditions are as follows:
an Agilent high performance liquid chromatograph;
a chromatographic column: shimadzu GL ODS C18;
flow rate: 0.85 mL/min;
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
sample concentration: 2 mg/ml;
diluent agent: 0.01mol/L dipotassium hydrogen phosphate (pH = 7.0) solution: methanol =1: 1;
detection wavelength:
mobile phase A: a mixed solution of 0.5% of sodium heptanesulfonate, 0.1% of sodium lauryl sulfate, 1.2% of tetrabutylammonium hydrogen sulfate (mass fraction), and 0.10% of boric acid (volume fraction).
Mobile phase B: methanol
TABLE 5 results of the experiment
As can be seen from table 5, the separation of each compound was good.
Example 4
The experimental conditions are as follows:
waters ultra high performance liquid chromatograph;
a chromatographic column: waters BEH C182.1X 100mm 1.7 μm;
flow rate: 0.2 mL/min;
column temperature: 30 ℃;
sample introduction amount: 2 mu L of the solution;
sample concentration: 3 mg/ml;
diluent agent: 0.01mol/L dipotassium hydrogen phosphate (pH = 7.0) solution: methanol =1: 1;
detection wavelength:
mobile phase A: 0.75% of sodium heptanesulfonate, 0.08% of sodium dodecyl sulfate, 1.5% of tetrabutylammonium hydrogen sulfate (mass fraction) and 0.15% of acetic acid (volume fraction).
Mobile phase B: methanol
TABLE 6 results of the experiment
As can be seen from table 6, the separation of each compound was good.
Example 5
The experimental conditions are as follows:
waters ultra high performance liquid chromatograph;
a chromatographic column: waters BEH C182.1X 100mm 1.7 μm;
flow rate: 0.2 mL/min;
column temperature: 30 ℃;
sample introduction amount: 2 mu L of the solution;
sample concentration: 3 mg/ml;
diluent agent: 0.01mol/L dipotassium hydrogen phosphate (pH = 7.0) solution: methanol =1: 1;
detection wavelength:
mobile phase A: 0.75% of sodium heptanesulfonate, 0.08% of sodium dodecyl sulfate, 1.5% of tetrabutylammonium hydrogen sulfate (mass fraction) and 0.15% of acetic acid (volume fraction).
Mobile phase B: methanol
TABLE 7 results of the experiment
As can be seen from table 7, the separation of each compound was good.
Example 6
The experimental conditions are as follows:
waters ultra high performance liquid chromatograph;
a chromatographic column: waters BEH C182.1X 100mm 1.7 μm;
flow rate: 0.2 mL/min;
column temperature: 30 ℃;
sample introduction amount: 2 mu L of the solution;
sample concentration: 3 mg/ml;
diluent agent: 0.01mol/L dipotassium hydrogen phosphate (pH = 7.0) solution: methanol =1: 1;
detection wavelength:
mobile phase A: 0.75% of sodium heptanesulfonate, 0.08% of sodium dodecyl sulfate, 1.5% of tetrabutylammonium hydrogen sulfate (mass fraction) and 0.15% of formic acid (volume fraction).
Mobile phase B: methanol
TABLE 8 results of the experiment
As can be seen from table 8, the separation of each compound was good.
Example 7
First, instruments and conditions
Waters ultra high performance liquid chromatograph, Milli-Q ultra pure water machine, Mettler XS205DU electronic balance, ultrasonic cleaner, vacuum filtration device, chromatographic column: waters BEH C182.1X 100mm 1.7 μm; flow rate: 0.2 mL/min;
column temperature: 30 ℃;
sample introduction amount: 2 mu L of the solution;
sample concentration: 1 mg/ml;
diluent agent: 0.01mol/L dipotassium hydrogen phosphate (pH = 7.0) solution: methanol =1: 1;
detection wavelength:
mobile phase A: a mixed solution of 0.50% of sodium heptanesulfonate, 0.10% of sodium lauryl sulfate, 1.2% of tetrabutylammonium hydrogen sulfate (mass fraction) and 0.10% of acetic acid (volume fraction).
Mobile phase B: methanol
Second, the experimental procedure
1. Preparing a solution:
impurity control stock solution: taking a proper amount of each of a p-hydroxyphenylacetic acid reference substance, a p-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester reference substance, a p-guanidinobenzoic acid reference substance, an impurity A reference substance and an impurity B reference substance, precisely weighing, dissolving by using a diluent, and quantitatively diluting into a solution containing about 10 mu g of each impurity per 1 ml.
Linear solution: and respectively preparing a p-hydroxyphenylacetic acid reference substance, a p-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester reference substance, a p-guanidinobenzoic acid reference substance, an impurity A reference substance, an impurity B reference substance and a camostat mesylate reference diluent into a mixed solution with the LOQ concentration of 3 mu g/ml.
System applicability solution: 10mg of the camostat mesylate reference substance is precisely weighed and placed in a 10ml measuring flask, and the reference substance is dissolved by the impurity stock solution and the volume is determined.
Camostat mesylate localization solution: taking a proper amount of the camostat mesylate reference substance, precisely weighing, dissolving by using a diluent, and quantitatively diluting to prepare a solution containing 1.0mg of camostat mesylate per 1 ml.
P-hydroxyphenylacetic acid positioning solution: taking a proper amount of p-hydroxyphenylacetic acid reference substance, precisely weighing, dissolving with diluent, and quantitatively diluting to obtain a solution containing 10 μ g of p-hydroxyphenylacetic acid per 1 ml.
P-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester positioning solution: taking a proper amount of p-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester reference substance, precisely weighing, dissolving by using a diluent, and quantitatively diluting to prepare a solution containing 10 mu g of p-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester per 1 ml.
P-guanidinobenzoic acid positioning solution: taking a proper amount of p-guanidinobenzoic acid reference substance, precisely weighing, dissolving with diluent, and quantitatively diluting to obtain solution containing 10 μ g per 1 ml.
Impurity a localization solution: taking a proper amount of the reference substance of the impurity A, precisely weighing, dissolving by using a diluent, and quantitatively diluting to prepare a solution containing 10 mu g of the impurity A per 1 ml.
Impurity B localization solution: taking a proper amount of reference substance of the impurity B, precisely weighing, dissolving by using a diluent, and quantitatively diluting to prepare a solution containing 10 mu g of the impurity B per 1 ml.
Mixing the solution: the same applies to the solution.
Control solution: precisely measuring 0.1ml of impurity reference substance stock solution, placing the impurity reference substance stock solution into a 100ml measuring flask, dissolving the impurity reference substance stock solution by using a diluent, and fixing the volume.
Test solution: an appropriate amount of the product is precisely weighed, dissolved by a diluent and quantitatively diluted to prepare 1.0mg of camostat mesylate solution per 1ml, and two parts are prepared in parallel.
0.1% self-control solution: precisely measuring 0.1ml of test solution, placing the test solution in a 100ml measuring flask, dissolving the test solution by using a diluent and fixing the volume.
2. Methodology validation
2.1 specificity test
And (3) sequentially injecting 2 mu L of each of a diluent positioning solution, a p-hydroxyphenylacetic acid-N, N-dimethylcarbamyl methyl ester positioning solution, a p-guanidinobenzoic acid positioning solution, an impurity A positioning solution, an impurity B positioning solution, a camostat mesylate positioning solution and a mixed solution into a liquid chromatogram, and recording a chromatogram. Results are shown in Table 9 and FIGS. 1 to 8.
TABLE 9 results of the specificity test
2.2 Linear sum Range, detection limits and quantitation limits
TABLE 10 results of the linearity and Range test
TABLE 11 detection limit and quantitation limit test results
2.3 sample determination
P-hydroxyphenylacetic acid and p-hydroxyphenylacetic acid-N, N-dimethyl carbamyl methyl ester are measured by adopting a reference external standard method, and the guanylbenzoic acid, the impurity A and the impurity B are measured by adopting a self-reference method of adding relative influence factors.
2.4 conclusion
The detection method for related substances of the camostat mesylate, disclosed by the invention, has the advantages of strong specificity, high sensitivity, accurate quantification and capability of well performing quality control on related substances of the camostat mesylate.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. A detection method of related substances of camostat mesylate is characterized by comprising the following steps: the detection method is liquid chromatography, and the chromatographic conditions are as follows:
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column;
the mobile phase A is: the ion pair solution, the mobile phase B is one or two of acetonitrile, methanol and ethanol;
the diluent is: pure water or salt solution, or a mixed solution of pure water or salt solution and one or two of methanol, acetonitrile and ethanol;
the ion pair solution refers to: adding a certain amount of acid into a mixed solution of sodium heptanesulfonate, sodium dodecyl sulfate and tetrabutylammonium hydrogen sulfate;
the acid refers to: any one or more of acetic acid, phosphoric acid, methanesulfonic acid, boric acid, and formic acid;
the salt solution is one or a mixture of two of disodium hydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and ammonium formate;
the detector is one of an ultraviolet detector and a diode array detector; the detection wavelength of the detector is 210 nm-280 nm;
the chromatographic column is selected from: kromasil C18, ODS-C18, Thermo Syncronis C18, Thermo Hypersil GOLD C18, Waters symmetry C18, Waters RP C18, Agilent XDB C18, Agilent eclipse plus C18, Shimadzu GL ODS C18, Waters BEH C18; the flow rate of the mobile phase is 0.8-1.2 ml/min; the column temperature is 30-40 ℃;
carrying out sample injection analysis according to a certain amount of sample concentration, wherein the sample concentration is 0.5-3.0 mg/ml;
the mobile phase A: and carrying out gradient elution on the mobile phase B, wherein the elution ratio is as follows:
2. the detection method according to claim 1, characterized in that: the ion pair solution of the mobile phase A comprises 0.5-1.5% of sodium heptanesulfonate, 0.05-0.5% of sodium dodecyl sulfate and 1-4% of tetrabutylammonium hydrogen sulfate, wherein the percentages are mass percent and 0.05-1.0% of acetic acid, and are volume percent; the mobile phase B is methanol.
3. The detection method according to claim 2, characterized in that: the ion pair solution of mobile phase a contained 0.5% sodium heptanesulfonate, 0.1% sodium lauryl sulfate, 2% tetrabutylammonium hydrogen sulfate, and 0.1% acetic acid.
4. The detection method according to claim 1, characterized in that: the diluent is a mixed solution of 0.005-0.1 mol/L dipotassium hydrogen phosphate solution and methanol, wherein the pH value of the mixed solution is 5.0-7.5; the content of the methanol in the mixed solution is 10% -60%, and the percentage is volume percentage.
5. The detection method according to claim 4, characterized in that: the pH value of the potassium dihydrogen phosphate solution is 7.0, and the concentration is 0.01 mol/L.
6. The detection method according to claim 1, characterized in that: the detector is a diode array detector; the detection wavelengths are as follows:
7. the detection method according to claim 1, characterized in that: the chromatographic column is Waters BEH C18.
8. The use of the assay of claim 1 for the detection of starting material related substances in a camostat mesylate synthesis process.
9. The use of the method of claim 1 for detecting the residual amount of 4-dimethylaminopyridine as a catalyst for a synthesis process and the residual amounts of acetone and DMF as purification solvents.
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