Method for detecting 5-Isoquinolinesulfonic acid in fasudil hydrochloride

A technology of ethyl isoquinolinesulfonate and fasudil hydrochloride, which is applied in the field of detection of ethyl 5-isoquinolinesulfonate in fasudil hydrochloride, can solve the problem of 5-isoquinolinesulfonate Ethyl acetate detection method, ineffective detection, long running time and other problems, to achieve the effect of high detection sensitivity, good for safety control, and short running time

Active Publication Date: 2019-02-22

KPC PHARM INC

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Problems solved by technology

The quantitative concentration of this method is 0.337 μg/ml, and the detection limit concentration is 0.084 μg/ml, which is still high, and the sensitivity is not enough, and it cannot effectively detect methyl 5-isoquinolinesulfonate at the ppm level in ...

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Abstract

The invention belongs to the field of medicinal chemistry analysis and particularly relates to a method for detecting 5-Isoquinolinesulfonic acid in fasudil hydrochloride. The method is that a high-performance liquid chromatography-mass spectrum system is adopted to detect a 5-Isoquinolinesulfonic acid reference substance solution and a fasudil hydrochloride test sample solution. According to thedetecting method, the high performance liquid chromatography is firstly adopted for component separation, the position of a chromatographic peak of the 5-Isoquinolinesulfonic acid in the fasudil hydrochloride test sample solution can be determined under the chromatographic condition, the chromatographic peak of the 5-Isoquinolinesulfonic acid is effectively separated from chromatographic peak of adjacent other components, and the operation time is short; then, the mass spectrum condition is adopted to quantify a ternary quadrupole rod mass spectrum, ppm level of 5-Isoquinolinesulfonic acid canbe detected, and the limit of quantitation can reach ng/ml. Therefore, the 5-Isoquinolinesulfonic acid in fasudil hydrochloride can be effectively controlled.

Application Domain

Component separation

Technology Topic

DrugChemistry +9

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Examples

Experimental program(13)

Example Embodiment

[0041] Example 1

[0042] 1. Instruments and materials

[0043] 1. Main instruments

[0044] Mass spectrometer: AB SCIEX company, model API 4000.

[0045] Liquid chromatograph: Shimadzu company, model LC20AD.

[0046] Electronic balance: METTLER company, XP205 type.

[0047] 2. Experimental materials

[0048] Ethyl 5-isoquinolinesulfonate reference substance: Provided by Kunyao Group Co., Ltd., with a purity of 97.62%.

[0049] Fasudil Hydrochloride API: Kunyao Group Co., Ltd., batch number 201201-01.

[0050] 2. Experimental method

[0051] 1. Solution preparation

[0052] 1.1 0.1% formic acid-water solution: accurately pipette 1ml of formic acid into 1000ml of water and mix well.

[0053] 1.2 Diluent: Measure 300ml of water and 700ml of acetonitrile, and mix well.

[0054] 1.3 Reference substance stock solution (50ng/ml): Take about 2mg of 5-isoquinoline sulfonate reference substance, accurately weigh it, and place it in a 200ml measuring flask, dissolve it with diluent and dilute to the mark, shake it well; Take 1.0ml, put it in a 200ml measuring flask, dilute to the mark with diluent, shake well, and use it as a reference substance stock solution.

[0055] 1.4 Reference substance solution (7.5ng/ml): Precisely pipette 3.0ml reference substance stock solution, place it in a 20ml volumetric flask, dilute to the mark with diluent, shake well, and use as the reference substance solution.

[0056] 1.5 Sensitivity solution (0.5ng/ml): Precisely pipette 1.0ml of the reference substance stock solution, place it in a 100ml measuring flask, dilute to the mark with diluent, and shake it up as a sensitivity solution.

[0057] 1.6 Prepare the specific solution: Take about 10mg of the test product of fasudil hydrochloride, accurately weigh it, place it in a 20ml measuring bottle, and accurately add 3.0ml of the reference substance stock solution, dilute to the mark with a diluent, and shake it as the specificity Solution.

[0058] 1.7 Test solution (0.5mg/ml): Take 10 mg of fasudil hydrochloride raw material, accurately weigh it, and place it in a 20ml measuring flask, dissolve it with a diluent and dilute to the mark, shake it up, as the test solution.

[0059] 2. Method parameters

[0060] The method parameters are shown in Table 1.

[0061] Table 1

[0062]

[0063] 3. System suitability test

[0064] 3.1 Take a blank solvent (diluent) into the liquid chromatograph, record the chromatogram (see figure 1 ). The blank solvent has no interference at the retention time of ethyl 5-isoquinolinesulfonate.

[0065] 3.2 Inject the specific solution into the liquid chromatograph and record the chromatogram (see figure 2 ). The signal-to-noise ratio of ethyl 5-isoquinoline sulfonate is greater than 10.

[0066] 3.3 Inject the reference solution into the liquid chromatograph and record the chromatogram (see image 3 ). The RSD of the peak area of ethyl 5-isoquinolinesulfonate in the solution for 5 consecutive points was 5.1%, as shown in Table 2.

[0067] Table 2. System suitability test results-reference solution

[0068]

[0069] Conclusion: The applicability of the system meets the requirements, and the specificity of this method is good.

[0070] 4. Linear range

[0071] Linear stock solution (50ng/ml): Take the reference substance stock solution as the linear stock solution.

[0072] Prepare the linear test solution according to Table 3, inject it into the chromatograph, record the chromatogram, perform linear regression analysis with the concentration as the abscissa and the peak area as the ordinate to obtain the linear regression equation, and obtain the linear correlation coefficient r and slope. The specific results are shown in the table 4.

[0073] Table 3. Preparation of linear test solution

[0074] Level

[0075] Table 4. Linearity results of ethyl 5-isoquinoline sulfonate

[0076]

[0077] The results showed that the linear equation of ethyl 5-isoquinoline sulfonate in the concentration range of 1.03ng/ml to 10.25ng/ml was y=10660x-73400, the correlation coefficient r was 0.9950, and the slope was 10660.

[0078] Conclusion: Ethyl 5-isoquinoline sulfonate has a good linear relationship in the concentration range of 1.03ng/ml to 10.25ng/ml.

[0079] 5. Limit of quantification and limit of detection

[0080] Limit of quantification solution: Take the linear solution of 2ppm under item 4 as the limit of quantification solution, accurately pipette 5.0ml into a 10ml volumetric flask, dilute to the mark with diluent, shake well, as the limit of quantification solution.

[0081] Detection limit solution: Precisely pipette 3.0ml of the limit of quantification solution into a 10ml measuring flask, dilute to the mark with a diluent, and shake it up as the detection limit solution.

[0082] Take the above solution into the liquid chromatography-mass spectrometer, and record the chromatogram. The results are shown in Table 5.

[0083] Table 5. Results of limit of quantification and detection limit of ethyl 5-isoquinoline sulfonate

[0084]

[0085] The results showed that the limit of quantification of ethyl 5-isoquinolinesulfonate was 0.5127ng/ml, the concentration level was 1ppm, the RSD of the peak area of the continuous 6-shot solution was 3.1%, and the signal-to-noise ratio was greater than 10; 5-isoquine The detection limit concentration of ethyl morpholinosulfonate is 0.1538ng/ml, the concentration level is 0.3ppm, and the signal-to-noise ratio is 6.7.

[0086] Conclusion: It shows that the detection sensitivity of this method can meet the requirements.

[0087] 6. Accuracy and precision

[0088] Preparing accuracy test solution (5ppm): Take about 10mg of fasudil hydrochloride test substance, accurately weigh it, and place it in a 20ml measuring bottle. Add 1.0ml reference substance stock solution accurately, dilute to the mark with diluent, and shake Evenly, as the accuracy test solution, prepare 6 copies in parallel.

[0089] Inject the test solution, reference solution, and accuracy test solution into the liquid chromatography-mass spectrometer, record the chromatogram, and calculate the recovery rate. The results are shown in Table 6.

[0090] Table 6. Accuracy & precision results of fasudil hydrochloride genotoxic impurity 5-isoquinoline sulfonate

[0091]

[0092] The results showed that the recovery rate of ethyl 5-isoquinolinesulfonate in the 6 spiked samples was in the range of 89.9% to 101.5%; the average recovery rate was 95.4%, and the RSD was 4.4%.

[0093] Conclusion: It shows that the accuracy and precision of this method are good.

[0094] 7. Solution stability

[0095] Take the reference solution and the specific solution, test on day 0, and calculate the content of ethyl 5-isoquinoline sulfonate in the specific solution according to the method. Divide the reference solution and the specific solution into two parts, store one at room temperature and the other at 4°C. After 1 day, the sample will be injected for testing, and the reference solution will be calculated based on the newly prepared reference solution that day Concentration and specific content of ethyl 5-isoquinoline sulfonate in the solution. Compare with the 0-day data and calculate the relative deviation. The results are shown in Table 7 below.

[0096] Table 7. Solution stability test results

[0097]

[0098] The results showed that the reference solution was stored at room temperature and 4°C for 1 day, and the relative deviation of the concentration of ethyl 5-isoquinoline sulfonate was less than 10% compared with the 0 day data. The specific solution was stored at room temperature and 4°C for 1 day, and the relative deviation of ethyl 5-isoquinoline sulfonate content was less than 10% compared with the 0 day data.

[0099] Conclusion: The reference solution and sample solution are stable within one day at room temperature and 4°C.

Example Embodiment

[0100] Example 2. Quality analysis of fasudil hydrochloride by the method of the present invention

[0101] The raw material of Fasudil Hydrochloride comes from Kunyao Group Co., Ltd. There are 3 batches with batch numbers 201201-01, 201201-02, 201201-03.

[0102] Preparation of test solution (0.5mg/ml): Take 10mg of fasudil hydrochloride crude drug, accurately weigh it, and place it in a 20ml measuring flask, dissolve it with diluent and dilute to the mark, shake it up, as the test solution .

[0103] Take the above-mentioned sample solution and inject it into high performance liquid chromatography with triple quadrupole mass spectrometer, and record the chromatogram (for the chart of batch 201201-01, see Figure 4 , 201201-02 batch map and Figure 4 Similar, see the map of batch 201201-03 Figure 5 ), calculate the content of genotoxic impurity 5-isoquinoline sulfonate ethyl ester in the test product according to the linear equation under item 4. The results are shown in Table 8.

[0104] Table 8. Determination results of fasudil hydrochloride genotoxic impurity 5-isoquinoline sulfonate ethyl ester

[0105] Sample lot number

[0106] The results showed that in the three batches of samples, the content of ethyl 5-isoquinoline sulfonate was less than 15 ppm.

Example Embodiment

[0107] Example 3

[0108] This embodiment is the same as Example 1, except that the concentration of the fasudil hydrochloride test solution is 0.4 mg/ml, and the concentration of the 5-isoquinoline sulfonate ethyl reference solution is 0.1 ng/ml.

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PUM

Property	Measurement	Unit
Length	50.0	mm
The inside diameter of	4.6	mm
Particle size	5.0	µm
tensile		MPa
Particle size		Pa
strength	10

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Method for detecting 5-Isoquinolinesulfonic acid in fasudil hydrochloride

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