CN109374811B - Method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride - Google Patents
Method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride Download PDFInfo
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Abstract
The invention belongs to the field of pharmaceutical chemistry analysis, and particularly relates to a detection method of 5-isoquinoline ethyl sulfonate in fasudil hydrochloride. The method is characterized in that a high performance liquid chromatography-mass spectrometry system is adopted to detect a 5-isoquinoline ethyl sulfonate reference solution and a fasudil hydrochloride test solution respectively. The detection method comprises the steps of firstly, separating components by adopting a high performance liquid chromatography, wherein the chromatographic condition can determine the position of a chromatographic peak of 5-isoquinoline ethyl sulfonate in a fasudil hydrochloride test solution, the chromatographic peak of the 5-isoquinoline ethyl sulfonate is effectively separated from chromatographic peaks of other adjacent components, and the running time is short; then, the triple quadrupole mass spectrometry is adopted for quantification under the mass spectrometry condition, the 5-isoquinoline ethyl sulfonate with ppm level can be detected, and the quantification limit can reach the ng/ml level, so that the 5-isoquinoline ethyl sulfonate in the fasudil hydrochloride can be effectively controlled.
Description
Technical Field
The invention belongs to the field of pharmaceutical chemistry analysis, and particularly relates to a detection method of 5-isoquinoline ethyl sulfonate in fasudil hydrochloride.
Background
Fasudil hydrochloride is a novel isoquinoline sulfonamide derivative, can expand blood vessels by increasing the activity of myosin light chain phosphatase, reduce the tension of endothelial cells, improve the microcirculation of brain tissues, protect ischemic brain tissues, antagonize inflammatory factors, protect nerves against apoptosis and promote nerve regeneration. Fasudil hydrochloride and injection thereof are marketed in China in 2004 and are mainly used for improving ischemic cerebrovascular disease symptoms caused by cerebral vasospasm and the like after subarachnoid hemorrhage.
Impurity control is one of the important contents of medicine quality control, and whether impurities in medicines can be comprehensively and accurately controlled is directly related to the controllability and safety of the quality of the medicines. Therefore, the research on impurities is carried out in a standard way, and the impurities are controlled within a safe and reasonable limit range, which has important significance on the clinical safety of the medicine. A synthetic route of fasudil hydrochloride can generate a plurality of impurities, 5-isoquinoline ethyl sulfonate is an impurity with genotoxic effect, and can be transferred to a finished product of fasudil hydrochloride in the production process, and corresponding control and detection are needed to be carried out to ensure the safety and reliability of the product. Genotoxic impurities are generally allowed to be contained within a very low limit, and at the ppm level, the detection limit and the quantification limit are difficult to reach by common detection methods. At present, the detection of impurities in fasudil hydrochloride is generally the detection of common substances, and the detection of 5-isoquinoline ethyl sulfonate is not found. In the related art, the applicant has developed a method for determining genotoxic impurities in fasudil hydrochloride by an HPLC method (li kaifeng et al, determination of genotoxic impurities in fasudil hydrochloride by an HPLC method, strait pharmacology, 2018, 02 th stage), which employs a high performance liquid chromatography, a chromatographic column: thermo Scientific HypersilGOLS C8(4.6 × 150mm, 3 μm), mobile phase: 0.1% formic acid solution-acetonitrile (98: 2) isocratic elution; detection wavelength: 275nm, flow rate: 0.9m L. min-1(ii) a Sample introduction amount: 20 mu L of the solution; column temperature: at 30 ℃. The quantitative concentration of the method is 0.337 mug/ml, the detection limit concentration is 0.084 mug/ml, the method is still high, the sensitivity is insufficient, and the method cannot effectively detect ppm level 5-isoquinoline methyl sulfonate in fasudil hydrochloride. The method is not a detection method aiming at 5-isoquinoline ethyl sulfonate, and has long running time and insufficient sensitivity. No report about a detection method of 5-isoquinoline ethyl sulfonate is found in the prior art.
The present invention has been made in view of this situation.
Disclosure of Invention
The existing method for detecting 5-isoquinoline methyl sulfonate in fasudil hydrochloride has the quantitative concentration of 0.337 mug/ml and the detection limit concentration of 0.084 mug/ml, and can not effectively detect ppm level 5-isoquinoline methyl sulfonate in fasudil hydrochloride. The method is used for detecting 5-isoquinoline methyl sulfonate, and the detection for 5-isoquinoline ethyl sulfonate is not found. Aiming at the problem, the invention aims to provide a method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride, which has the advantages of very short operation time, higher sensitivity and capability of effectively controlling the 5-isoquinoline ethyl sulfonate in the fasudil hydrochloride, wherein both the quantitative concentration and the detection limit reach the ppm level.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride is characterized in that a high performance liquid chromatography-mass spectrometry system is adopted to respectively detect a 5-isoquinoline ethyl sulfonate reference solution and a fasudil hydrochloride test solution.
A synthetic route of fasudil hydrochloride can generate a plurality of impurities, 5-isoquinoline ethyl sulfonate is an impurity with genotoxic effect, and can be transferred to a finished product of fasudil hydrochloride in the production process, and corresponding control and detection are needed to be carried out to ensure the safety and reliability of the product. Genotoxic impurities are generally allowed to be contained within a very low limit, and at the ppm level, the detection limit and the quantification limit are difficult to reach by common detection methods.
Specifically, the method comprises the following steps:
1) taking a reference substance of 5-isoquinoline ethyl sulfonate, preparing a reference substance solution, and detecting by using a high performance liquid chromatography-mass spectrometry system to obtain a standard curve;
2) taking a fasudil hydrochloride sample to be detected, preparing a sample solution, and detecting by using a high performance liquid chromatography-mass spectrometry system;
3) and calculating the peak area of the 5-isoquinoline ethyl sulfonate in the test solution, substituting the peak area into a standard curve equation, and calculating the content of the 5-isoquinoline ethyl sulfonate in the fasudil hydrochloride.
Furthermore, in the high performance liquid chromatography-mass spectrometry system, a gradient elution mode is adopted in the high performance liquid chromatography condition; the mobile phase is A: 0.1% formic acid-water solution, B: 0.1% formic acid-acetonitrile.
Preferably, the gradient elution is performed using the following elution procedure:
0.01min, the volume ratio of the mobile phase A phase is 98%, and the volume ratio of the mobile phase B phase is 2%;
0.1min, the volume ratio of the mobile phase A phase is 98%, and the volume ratio of the mobile phase B phase is 2%;
1.3min, the volume ratio of the mobile phase A phase is 2%, and the volume ratio of the mobile phase B phase is 98%;
1.79min, the volume ratio of the mobile phase A is 2%, and the volume ratio of the mobile phase B is 98%;
1.8min, wherein the volume ratio of the mobile phase A is 98%, and the volume ratio of the mobile phase B is 2%;
2.3min, the volume ratio of the mobile phase A phase is 98%, and the volume ratio of the mobile phase B phase is 2%.
Further, in the high performance liquid chromatography-mass spectrometry system, the mass spectrum is a triple quadrupole mass spectrum, and the mass spectrometry conditions are as follows: the ion mode is ESI +; first mass spectrum ion Q1 is m/z 238.2 (parent ion); secondary mass spectrum ion Q3 was m/z 210.2 (quantitation ion).
Further, in the hplc-ms system, the mass spectrum parameters are: the de-clustering voltage DP is 80V, the atomizing gas is 50psi, the heating gas is 50psi, the electrospray voltage is 5500V, the curtain gas is 20psi, the ionization temperature is 550 ℃, and the collision energy CE is 24eV, wherein the related parameters float up and down by 50%, preferably 10%, and more preferably 5%; most preferred mass spectral parameters are: the de-clustering voltage DP was 80V, the atomizing gas was 50psi, the heating gas was 50psi, the electrospray voltage was 5500V, the curtain gas was 20psi, the ionization temperature was 550 deg.C, and the collision energy CE was 24 eV.
Furthermore, the concentration of the fasudil hydrochloride test solution can be 0.01-2mg/ml, preferably 0.05-1.5mg/ml, more preferably 0.1-1.0mg/ml, still more preferably 0.4-0.6mg/ml, and most preferably 0.5 mg/ml.
Further, the solvent for preparing the fasudil hydrochloride test solution is water-acetonitrile, wherein the volume ratio of water to acetonitrile is 3: 7.
preparing fasudil hydrochloride test solution by adopting a method that the volume ratio is 3: the water-acetonitrile of 7 is used as a solvent, fasudil hydrochloride can be effectively dissolved, the solvent has no interference at the retention time of the 5-isoquinoline ethyl sulfonate, and the signal-to-noise ratio of the 5-isoquinoline ethyl sulfonate is greater than 10.
Further, the concentration of the ethyl 5-isoquinolinesulfonate control solution is 0.1-50ng/ml, preferably 1-20ng/ml, more preferably 5-10ng/ml, and still more preferably 7.5 ng/ml.
Further, the solvent of the ethyl 5-isoquinolinesulfonate control solution is water-acetonitrile, wherein the volume ratio of water to acetonitrile is 3: 7.
further, in the HPLC-MS system, the column used in the HPLC conditions is a reversed phase column, preferably octanesilane bonded silica, more preferably Diamonsil C8, 150mm in length, 4.6mm in inner diameter, and 3 μm in filler particle size.
Furthermore, in the high performance liquid chromatography-mass spectrometry system, the flow rate is 0.9ml/min, the sample injection volume is 10 mul, and the running time is 2.3min under the high performance liquid chromatography condition.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
the detection method comprises the steps of firstly, separating components by adopting a high performance liquid chromatography, wherein the chromatographic condition can determine the position of a chromatographic peak of 5-isoquinoline ethyl sulfonate in a fasudil hydrochloride test solution, the chromatographic peak of the 5-isoquinoline ethyl sulfonate is effectively separated from chromatographic peaks of other adjacent components, and the running time is short; then, the triple quadrupole mass spectrometry is adopted for quantification under the mass spectrometry condition, the 5-isoquinoline ethyl sulfonate with ppm level can be detected, and the quantification limit can reach the ng/ml level, so that the 5-isoquinoline ethyl sulfonate in the fasudil hydrochloride can be effectively controlled.
The method disclosed by the invention is stable and reliable, short in time consumption, good in specificity and high in detection sensitivity, the detection limit concentration of the 5-isoquinoline ethyl sulfonate can reach 0.1538ng/ml, and the genotoxic component 5-isoquinoline ethyl sulfonate in fasudil hydrochloride can be better controlled, so that the safety control of the medicine is facilitated.
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
FIG. 1 is an air-white solvent chromatogram in example 1 of the present invention;
FIG. 2 is a proprietary solution chromatogram of example 1 of the present invention;
FIG. 3 is a chromatogram of a control solution in example 1 of the present invention;
FIG. 4 is a chromatogram of a test solution of sample 201201-01 in example 2 according to the present invention;
FIG. 5 is a chromatogram of a sample solution 201201-03 of example 2 according to the present invention.
It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1
Instrument and material
1. Main instrument
Mass spectrometry: AB SCIEX, Inc., model API 4000.
Liquid chromatograph: shimadzu corporation, model LC20 AD.
An electronic balance: METTLER corporation, XP205 type.
2. Experimental Material
5-isoquinoline ethyl sulfonate control: supplied by Kunzi group, Inc., with a purity of 97.62%.
Fasudil hydrochloride bulk drug: kun Yao group, Inc., lot number 201201-01.
Second, Experimental methods
1. Solution preparation
1.10.1% formic acid-water solution: precisely transferring 1ml of formic acid into 1000ml of water, and uniformly mixing.
1.2 diluents: 300ml of water and 700ml of acetonitrile are weighed and mixed evenly.
1.3 control stock (50 ng/ml): taking about 2mg of a 5-isoquinoline ethyl sulfonate reference substance, precisely weighing, placing in a 200ml measuring flask, dissolving and diluting to a scale with a diluent, and shaking up; precisely transferring 1.0ml, placing in a 200ml measuring flask, diluting to scale with diluent, and shaking to obtain reference stock solution.
1.4 control solution (7.5 ng/ml): precisely transferring 3.0ml of the reference stock solution, placing the reference stock solution in a 20ml measuring flask, diluting the reference stock solution to a scale with a diluent, and shaking up to obtain a reference solution.
1.5 sensitivity solution (0.5 ng/ml): precisely transferring 1.0ml of the reference stock solution, placing the reference stock solution in a 100ml measuring flask, diluting the reference stock solution to a scale with a diluent, and shaking up to obtain a sensitivity solution.
1.6 preparing a special solution: precisely weighing about 10mg of fasudil hydrochloride test sample, placing the test sample into a 20ml measuring flask, precisely adding 3.0ml of reference substance stock solution, diluting to scale with diluent, and shaking up to obtain a special solution.
1.7 test article solution (0.5 mg/ml): taking 10mg of fasudil hydrochloride raw material medicine, precisely weighing, placing in a 20ml measuring flask, dissolving and diluting to scale with diluent, shaking up, and taking as a test solution.
2. Method parameter
The process parameters are shown in Table 1.
TABLE 1
3. System suitability test
3.1 injecting blank solvent (diluent) into liquid chromatograph, recording chromatogram (see figure 1). The blank solvent did not interfere at the retention time of ethyl 5-isoquinolinesulfonate.
3.2 injecting the special solution into a liquid chromatograph, and recording a chromatogram (see figure 2). The signal-to-noise ratio of the 5-isoquinoline ethyl sulfonate is more than 10.
3.3 injecting the reference solution into a liquid chromatograph, and recording the chromatogram (see figure 3). The RSD of the peak area of 5-isoquinoline sulfonic acid ethyl ester in the continuous 5-pin control solution was 5.1%, as shown in Table 2.
TABLE 2 results of the System suitability test-control solution
And (4) conclusion: the system applicability meets the requirements, and the method has good specificity.
4. Linear range
Linear stock (50 ng/ml): the control stock solution was taken as the linear stock solution.
Preparing a linear test solution according to the table 3, injecting the linear test solution into a chromatograph, recording a chromatogram, performing linear regression analysis by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate to obtain a linear regression equation, and obtaining a linear correlation coefficient r and a slope, wherein specific results are shown in the table 4.
TABLE 3 Linear test solution preparation
| Level of | Concentration (ng/ml) | Linear stock solution (ml) | Final volume (ml) |
| L1(2ppm) | 1 | 1 | 50 |
| L2(5ppm) | 2.5 | 1 | 20 |
| L3(10ppm) | 5 | 2 | 20 |
| L4(15ppm) | 7.5 | 3 | 20 |
| L5(20ppm) | 10 | 2 | 10 |
TABLE 4, 5-Isoquinolinesulfonic acid Ethyl ester Linear results
The results show that: the linear equation of the 5-isoquinoline ethyl sulfonate in the concentration range of 1.03ng/ml to 10.25ng/ml is that y is 10660x-73400, the correlation coefficient r is 0.9950, and the slope is 10660.
And (4) conclusion: the 5-isoquinoline ethyl sulfonate has good linear relation in the concentration range of 1.03ng/ml to 10.25 ng/ml.
5. Quantitative limit and detection limit
Quantitative limiting solution: taking 2ppm linear solution under item 4 as quantitative limiting solution, precisely transferring 5.0ml into a 10ml measuring flask, diluting to scale with diluent, shaking up, and taking as quantitative limiting solution.
Detection limiting solution: precisely transferring 3.0ml of quantitative limiting solution into a 10ml measuring flask, diluting to scale with diluent, and shaking up to obtain detection limiting solution.
And (4) injecting the solution into a liquid chromatogram-mass spectrometer, and recording a chromatogram. The results are shown in Table 5.
TABLE 5 results of quantitation limit and detection limit of 5-isoquinolinesulfonic acid ethyl ester
The results show that: the quantitative limit concentration of the 5-isoquinoline ethyl sulfonate is 0.5127ng/ml, the concentration level is 1ppm, the RSD of the peak area of the continuous 6-needle solution is 3.1%, and the signal-to-noise ratio is more than 10; the detection limit concentration of the 5-isoquinoline ethyl sulfonate is 0.1538ng/ml, the concentration level is 0.3ppm, and the signal-to-noise ratio is 6.7.
And (4) conclusion: the detection sensitivity of the method can meet the requirement.
6. Accuracy and precision
Preparation of accuracy test solution (5 ppm): taking about 10mg of fasudil hydrochloride test sample, precisely weighing, placing in a 20ml measuring flask, precisely adding 1.0ml of reference substance stock solution, diluting to scale with diluent, shaking up, using as accuracy test sample solution, and preparing 6 parts in parallel.
And injecting the test solution, the reference solution and the accuracy test solution into a liquid chromatogram-mass spectrometer, recording a chromatogram, and calculating the recovery rate. The results are shown in Table 6.
TABLE 6 accuracy & precision results of fasudil hydrochloride genotoxic impurity ethyl 5-isoquinolinesulfonate
The results show that: the recovery rate of the 5-isoquinoline ethyl sulfonate in the 6 parts of the standard sample is within the range of 89.9-101.5%; the average recovery was 95.4% and the RSD was 4.4%.
And (4) conclusion: the method is shown to have good accuracy and precision.
7. Stability of solution
And taking the reference substance solution and the special solution, detecting for 0 day, and calculating the content of the 5-isoquinoline ethyl sulfonate in the special solution according to the method. And (3) packaging the reference substance solution and the special solution into two parts, storing one part at room temperature and the other part at 4 ℃, injecting a sample after 1 day for detection, and calculating the concentration of the reference substance solution and the content of the 5-isoquinoline ethyl sulfonate in the special solution according to the newly prepared reference solution on the same day. Relative deviations were calculated in comparison with day 0 data and the results are given in table 7 below.
TABLE 7 results of solution stability test
The results show that: the control solution was stored at room temperature and 4 ℃ for 1 day with a relative deviation of the ethyl 5-isoquinolinesulfonate concentration of less than 10% from the data for day 0. The specified solution was stored at room temperature and 4 ℃ for 1 day with a relative deviation of the ethyl 5-isoquinolinesulfonate content of less than 10% compared to the data for day 0.
And (4) conclusion: the control solution and the sample solution were stable at room temperature and 4 ℃ for 1 day.
Example 2 quality analysis of fasudil hydrochloride by the method of the invention
The fasudil hydrochloride raw material medicine is from Kunzhi group GmbH, 3 batches, and the batch numbers are 201201-01, 201201-02 and 201201-03 respectively.
Preparation of test solution (0.5 mg/ml): taking 10mg of fasudil hydrochloride raw material medicine, precisely weighing, placing in a 20ml measuring flask, dissolving and diluting to scale with diluent, shaking up, and taking as a test solution.
Injecting the test solution into a high performance liquid chromatography and a triple quadrupole mass spectrometer, recording a chromatogram (the chromatogram of 201201-01 batch is shown in figure 4, the chromatogram of 201201-02 batch is similar to figure 4, the chromatogram of 201201-03 batch is shown in figure 5), and calculating the content of the genotoxic impurity, namely 5-isoquinoline ethyl sulfonate, in the test solution according to a linear equation of 4 items. The results are shown in Table 8.
TABLE 8 determination results of fasudil hydrochloride genotoxic impurity, ethyl 5-isoquinolinesulfonate
| Sample batch number | Content (wt.) |
| 201201-01 | Not detected out |
| 201201-02 | Not detected out |
| 201201-03 | 0.8ppm |
The results show that: in 3 samples, the content of 5-isoquinoline ethyl sulfonate is less than 15 ppm.
Example 3
This example is the same as example 1 except that the concentration of fasudil hydrochloride test solution is 0.4mg/ml and the concentration of 5-isoquinoline ethyl sulfonate reference solution is 0.1 ng/ml.
Example 4
This example is the same as example 1 except that the concentration of fasudil hydrochloride test solution is 0.6mg/ml and the concentration of 5-isoquinoline ethyl sulfonate control solution is 50 ng/ml.
Example 5
This example is the same as example 1 except that the concentration of fasudil hydrochloride test solution is 0.1mg/ml and the concentration of 5-isoquinoline ethyl sulfonate control solution is 1 ng/ml.
Example 6
This example is the same as example 1 except that the concentration of fasudil hydrochloride test solution is 1.0mg/ml and the concentration of 5-isoquinoline ethyl sulfonate control solution is 20 ng/ml.
Example 7
This example is the same as example 1 except that the concentration of fasudil hydrochloride test solution is 0.05mg/ml and the concentration of 5-isoquinoline ethyl sulfonate control solution is 5 ng/ml.
Example 8
This example is the same as example 1 except that the concentration of fasudil hydrochloride test solution is 1.5mg/ml and the concentration of 5-isoquinoline ethyl sulfonate reference solution is 10 ng/ml.
Example 9
This example is the same as example 1 except that the concentration of fasudil hydrochloride test solution is 0.01mg/ml and the concentration of 5-isoquinoline ethyl sulfonate control solution is 7.5 ng/ml.
Example 10
This example is the same as example 1 except that the concentration of the fasudil hydrochloride test solution is 2 mg/ml.
Example 11
This example is the same as example 1, except that the mass spectral parameters are: the de-clustering voltage DP was 120V, the atomizing gas was 75psi, the heating gas was 75psi, the electrospray voltage 8250V, the curtain gas was 30psi, the ionization temperature was 825 deg.C, and the collision energy CE was 36 eV.
Example 12
This example is the same as example 1, except that the mass spectral parameters are: the de-clustering voltage DP was 40V, the atomizing gas 25psi, the heating gas 25psi, the electrospray voltage 2750V, the curtain gas 10psi, the ionization temperature 275 deg.C, and the collision energy CE 12 eV.
Example 13
This example is the same as example 1, except that the mass spectral parameters are: the de-clustering voltage DP was 80V, the atomizing gas was 45psi, the heating gas was 45psi, the electrospray voltage was 5125V, the curtain gas was 25psi, the ionization temperature was 500 deg.C, and the collision energy CE was 30 eV.
The methodological research on the specificity, the system applicability, the linear range, the quantitative limit, the detection limit, the accuracy, the precision and the solution stability of the method shows that the method is suitable for detecting the fasudil hydrochloride genotoxic impurity 5-isoquinoline ethyl sulfonate, and has the advantages of stability, reliability, short detection time and high sensitivity.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (12)
1. A method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride is characterized by comprising the following steps:
1) taking a reference substance of 5-isoquinoline ethyl sulfonate, preparing a reference substance solution, and detecting by using a high performance liquid chromatography-mass spectrometry system to obtain a standard curve;
2) taking a fasudil hydrochloride sample to be detected, preparing a sample solution, and detecting by using a high performance liquid chromatography-mass spectrometry system;
3) calculating the peak area of the 5-isoquinoline ethyl sulfonate in the test solution, substituting the peak area into a standard curve equation, and calculating the content of the 5-isoquinoline ethyl sulfonate in the fasudil hydrochloride;
wherein the chromatographic column used in the high performance liquid chromatography is reversed phase chromatographic column, is Diamonsil C8, has a length of 50mm and an inner diameter of 4.6mm, and the filler has a particle size of 5 μm; a gradient elution mode is adopted in the high performance liquid chromatography, and the mobile phase is A: 0.1% formic acid-water solution, B: 0.1% formic acid-acetonitrile solution; the gradient elution was performed using the following elution procedure:
0.01min, the volume ratio of the mobile phase A phase is 98%, and the volume ratio of the mobile phase B phase is 2%;
0.1min, the volume ratio of the mobile phase A phase is 98%, and the volume ratio of the mobile phase B phase is 2%;
1.3min, the volume ratio of the mobile phase A phase is 2%, and the volume ratio of the mobile phase B phase is 98%;
1.79min, the volume ratio of the mobile phase A is 2%, and the volume ratio of the mobile phase B is 98%;
1.8min, wherein the volume ratio of the mobile phase A is 98%, and the volume ratio of the mobile phase B is 2%;
2.3min, the volume ratio of the mobile phase A is 98%, and the volume ratio of the mobile phase B is 2%;
in the high performance liquid chromatography-mass spectrometry system, the mass spectrum is a triple quadrupole mass spectrum, and the mass spectrum conditions are as follows: the ion mode is ESI +; the primary mass spectrum ion Q1 is a parent ion m/z 238.2; the secondary mass spectrum ion Q3 is a quantitative ion m/z 210.2;
the mass spectrum parameters are as follows: the de-clustering voltage DP was 80V, the atomizing gas was 50psi, the heating gas was 50psi, the electrospray voltage was 5500V, the curtain gas was 20psi, the ionization temperature was 550 deg.C, and the collision energy CE was 24 eV.
2. The method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride according to claim 1, wherein the concentration of the fasudil hydrochloride test solution is 0.01-2 mg/ml.
3. The method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride according to claim 2, wherein the concentration of the fasudil hydrochloride test solution is 0.05-1.5 mg/ml.
4. The method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride according to claim 3, wherein the concentration of the fasudil hydrochloride test solution is 0.1-1.0 mg/ml.
5. The method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride according to claim 4, wherein the concentration of the fasudil hydrochloride test solution is 0.4-0.6 mg/ml.
6. The method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride according to claim 5, wherein the concentration of the fasudil hydrochloride test solution is 0.5 mg/ml.
7. The method for detecting 5-isoquinoline ethyl sulfonate in fasudil hydrochloride according to any one of claims 1 to 6, wherein the solvent for preparing the fasudil hydrochloride test solution is water-acetonitrile, wherein the volume ratio of water to acetonitrile is 3: 7.
8. the method for detecting ethyl 5-isoquinolinesulfonate in fasudil hydrochloride according to claim 1, wherein the concentration of the ethyl 5-isoquinolinesulfonate control solution is 0.1-50 ng/ml.
9. The method for detecting ethyl 5-isoquinolinesulfonate in fasudil hydrochloride according to claim 8, wherein the concentration of the ethyl 5-isoquinolinesulfonate control solution is 1-20 ng/ml.
10. The method for detecting ethyl 5-isoquinolinesulfonate in fasudil hydrochloride according to claim 9, wherein the concentration of the ethyl 5-isoquinolinesulfonate control solution is 5-10 ng/ml.
11. The method for detecting ethyl 5-isoquinolinesulfonate in fasudil hydrochloride according to claim 10, wherein the concentration of the ethyl 5-isoquinolinesulfonate control solution is 7.5 ng/ml.
12. The method for detecting ethyl 5-isoquinolinesulfonate in fasudil hydrochloride according to any one of claims 8-11, characterized in that the solvent of the ethyl 5-isoquinolinesulfonate control solution is water-acetonitrile, wherein the volume ratio of water to acetonitrile is 3: 7.
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