CN108459106B - Method for determining content of homopiperazine in fasudil hydrochloride

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Abstract

Description

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CN108459106B

Publication number: CN108459106B
Application number: CN201810388106.1A
Authority: CN
Inventors: 黄贤芳; 徐红兰; 顾凯; 朱子丰
Original assignee: Nanjing Milestone Pharma Co ltd
Current assignee: Nanjing Milestone Pharma Co ltd
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2021-01-26
Anticipated expiration: 2038-04-26
Also published as: CN108459106A

The invention provides a method for determining the content of homopiperazine in fasudil hydrochloride, and particularly relates to the technical field of drug detection, which comprises the following steps: s1, taking a homopiperazine reference substance, taking a 5% acetonitrile water solution as a solvent to prepare a homopiperazine reference substance solution, taking fasudil hydrochloride, taking a 5% acetonitrile water solution as a solvent to prepare a test sample solution; s2, preparing two high piperazine reference substance solutions in parallel, respectively obtaining peak areas of high piperazine in the reference substance solutions through detection of a UPLC-MS method, and calculating the ratio of the peak areas to the concentration of the reference substance solutions to obtain response factors of the high piperazine; s3, detecting the test solution by using a UPLC-MS method to obtain the peak area of the test solution, and calculating the content of the high piperazine in the test solution by using the response factor. The invention has the advantages of short detection time, good accuracy, high precision and good repeatability.

Method for determining content of homopiperazine in fasudil hydrochloride

Technical Field

The invention belongs to the technical field of drug detection, and particularly relates to a method for determining the content of homopiperazine in fasudil hydrochloride.

Background

Fasudil hydrochloride is a myosin light chain phosphorylase, RHO kinase inhibitor and novel intracellular calcium ion anticaking agent, can improve brain tissue microcirculation, effectively relieve cerebral vasospasm, protect ischemic brain tissue and promote nerve regeneration, is a novel medicament with wide pharmacological action, homopiperazine is an intermediate in the fasudil hydrochloride synthesis process, and since homopiperazine has no ultraviolet absorption and large polarity, efficient detection can not be carried out by adopting high performance liquid chromatography, ultraviolet spectrophotometry and ion chromatography are reported in the prior art to determine the content of homopiperazine in fasudil hydrochloride, but the existing detection method has low structural accuracy and is complicated to operate.

Therefore, a method for measuring the content of high piperazine in fasudil hydrochloride, which is simple, high in separation column efficiency, high in accuracy, simple, convenient and feasible, and can be used for quality control in the production process of fasudil hydrochloride, is urgently needed.

Disclosure of Invention

In order to solve the problems of low accuracy and complex operation of the conventional method for measuring the content of high piperazine in fasudil hydrochloride, the invention aims to provide the method for measuring the content of high piperazine in fasudil hydrochloride, and the method has the advantages of short detection time, good accuracy, high precision and good repeatability.

The invention provides the following technical scheme:

a method for determining the content of homopiperazine in fasudil hydrochloride comprises the following steps:

s1, taking a homopiperazine reference substance, taking a 5% acetonitrile water solution as a solvent to prepare a homopiperazine reference substance solution, taking fasudil hydrochloride, taking a 5% acetonitrile water solution as a solvent to prepare a test sample solution;

s2, preparing two high piperazine reference substance solutions in parallel, respectively obtaining peak areas of high piperazine in the reference substance solutions through detection of a UPLC-MS method, and calculating the ratio of the peak areas to the concentration of the reference substance solutions to obtain response factors of the high piperazine;

s3, detecting the test solution by using a UPLC-MS method to obtain the peak area of the test solution, and calculating the content of the high piperazine in the test solution by using the response factor.

Preferably, the chromatography conditions of the UPLC-MS method in the S2 step and the S3 step are: adopting a CSH C18 chromatographic column with the diameter of 2.1 multiplied by 100mm multiplied by 1.7 mu m and the column temperature is 35 ℃; the sample injection amount is 5 mu L; the detector is a mass spectrum detector, the ion source is ESI, the positive ion mode is adopted, and the ions are 101; taking 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate is 0.2 mL/min; the gradient elution procedure was: 0-1 min, wherein the volume percentage of the mobile phase B is 5%; 1-5 min, wherein the volume percentage of the mobile phase B is 5-90%; 5-5.1 min, wherein the volume percentage of the mobile phase B is from 90% to 5%; 5.1-7 min, and the volume percentage of the mobile phase B is 5%.

The invention has the beneficial effects that:

1. the UPLC-MS method adopted by the invention is used for measuring the content of homopiperazine, and as the compound homopiperazine is a degradation product of fasudil hydrochloride, the accuracy in the methodological verification of the gas chromatography cannot meet the detection requirement; and the homopiperazine has no ultraviolet response absorption, so the selection of the UPLC-MS method can accurately position the homopiperazine, and has strong specificity and high sensitivity.

2. The UPLC-MS method is adopted, the chromatographic time is short, the detection time is saved, the use of organic solvents is greatly reduced, the efficiency is improved, and the harm to the environment is reduced.

3. Under the condition of UPLC-MS provided by the invention, the high piperazine content in the test solution is determined by an external standard method, the operation is simple, and the methodological verification is carried out, and the experimental result shows that the detection method of the method has strong specificity, good accuracy, high precision and good repeatability, and meets the technical requirements of the quality standard research of the medicine.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a chromatogram of a control solution and a test solution of example 1;

FIG. 2 is a plot of the IPTG standard for the linear range investigation of example 2;

FIG. 3 is a specificity profile for the specificity test of example 5.

Detailed Description

Example 1

s1, taking a homopiperazine reference substance, taking a 5% acetonitrile water solution as a solvent, preparing a homopiperazine reference substance solution of 3 mu g/mL, taking fasudil hydrochloride, taking a 5% acetonitrile water solution as a solvent, and preparing a test solution of 1.5 mg/mL;

Specifically, the chromatographic conditions of the UPLC-MS method in the steps S2 and S3 are as follows: using CSH C18 chromatographic column (2.1 × 100mm, 1.7 μm) with a column temperature of 35 deg.C; the sample injection amount is 5 mu L; the detector is a mass spectrum detector, the ion source is ESI, the positive ion mode is adopted, and the ions are 101; taking 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate is 0.2 mL/min; the gradient elution procedure was: 0-1 min, wherein the volume percentage of the mobile phase B is 5%; 1-5 min, wherein the volume percentage of the mobile phase B is 5-90%; 5-5.1 min, wherein the volume percentage of the mobile phase B is from 90% to 5%; 5.1-7 min, and the volume percentage of the mobile phase B is 5%.

The experimental data for the gradient elution procedure are shown in table 1.

TABLE 1 gradient elution procedure

The gradient elution procedure provided in example 1 was used to perform UPLC-MS analysis on the control solution and the test solution, respectively, and the chromatograms obtained are shown in fig. 1, where reference 2 in the figure is the control solution and reference 3 is the test solution, and the retention time of homopiperazine is 0.896 min.

Example 2

Example 1 Linear Range examination of the assay methods provided

Dissolving 41.87mg of homopiperazine in a 25mL volumetric flask with 5% acetonitrile, fixing the volume to a scale, shaking up to obtain homopiperazine stock solution A; precisely transferring 1.0mL of the homopiperazine stock solution A into a 10mL volumetric flask, adding 5% acetonitrile for dilution, fixing the volume, and shaking up to obtain the homopiperazine stock solution B; precisely transferring 0.4mL of the homopiperazine stock solution B into a 10mL volumetric flask, adding 5% acetonitrile for dilution, fixing the volume, and shaking up to obtain the homopiperazine stock solution C; linear solutions of 0.3350. mu.g/mL, 0.6699. mu.g/mL, 1.3398. mu.g/mL, 2.0098. mu.g/mL, 2.6797. mu.g/mL, 3.3496. mu.g/mL, 5.0244. mu.g/mL, and 6.6992. mu.g/mL were prepared, labeled LOQ, 20%, 40%, 60%, 80%, 100%, 150%, and 200%, respectively.

200% -L (6.6992 μ g/mL): homopiperazine stock solution C; 150% -L (5.0244 μ g/mL): precisely transferring 0.75mL of homopiperazine stock solution C into a 2mL sample injection vial, adding 0.25mL of 5% acetonitrile for dilution, and shaking up; 100% -L (3.3496 μ g/mL): precisely transferring 0.5mL of homopiperazine stock solution C into a 2mL sample injection vial, adding 0.5mL of 5% acetonitrile for dilution, and shaking up; 80% -L (2.6797 μ g/mL): precisely transferring 0.4mL of homopiperazine stock solution C into a 2mL sample injection vial, adding 0.6mL of 5% acetonitrile for dilution, and shaking up; 60% -L (2.0098 μ g/mL): precisely transferring 0.3mL of homopiperazine stock solution C into a 2mL sample injection vial, adding 0.7mL of 5% acetonitrile for dilution, and shaking up; 40% -L (1.3398 μ g/mL): precisely transferring 0.2mL of homopiperazine stock solution C into a 2mL sample injection vial, adding 0.8mL of 5% acetonitrile for dilution, and shaking up; 20% -L (0.6699 μ g/mL): precisely transferring 0.1mL of homopiperazine stock solution C into a 2mL sample injection vial, adding 0.9mL of 5% acetonitrile for dilution, and shaking up; LOQ-L (0.3350. mu.g/mL): precisely transferring 0.05mL of the homopiperazine stock solution C into a 2mL sample injection vial, adding 0.95mL of 5% acetonitrile for dilution, and shaking up.

According to the determination method provided in example 1, samples are sequentially injected for determination, a standard curve is drawn by taking the peak area as the ordinate and the concentration of the reference solution as the abscissa (μ g/mL), the result is shown in table 2, and the drawn standard curve of high piperazine is shown in fig. 2.

TABLE 2 standard curve for homopiperazine

Example 3

Sample collection test for the measurement methods provided in example 1

Analyzing the accuracy of the determination method provided in example 1 by a sample adding and recycling method, taking 15mg of fasudil hydrochloride, precisely weighing, placing in a 10mL volumetric flask, paralleling 11 parts, taking 2 parts of the fasudil hydrochloride, and determining the content of high piperazine in the sample; taking 3 parts of the solution, respectively adding 0.1mL of homopiperazine stock solution B, adding 5% acetonitrile, fixing the volume, and uniformly mixing to obtain a solution with the standard addition recovery rate of 50%; taking 3 parts of the solution, respectively adding 0.2mL of homopiperazine stock solution B, adding 5% acetonitrile, fixing the volume and uniformly mixing to obtain a solution with the standard addition recovery rate of 100%; taking 3 parts of the piperazine solution, adding 0.3mL of the homopiperazine stock solution B, adding 5% of acetonitrile to the homopiperazine stock solution B to fix the volume, uniformly mixing the volume and the acetonitrile to obtain a 150% standard recovery rate solution, and measuring the standard recovery rate solution by the UPLC-MS method in example 1, wherein the results are shown in Table 3, and show that the high piperazine recovery rate is 93-119%, and the 9-pin RSD recovery rate is 9%, which indicates that the measuring method provided in example 1 has good accuracy.

TABLE 3 high piperazine recovery results

Example 4

Precision test of the measurement method provided in example 1

Analyzing the precision of the determination method provided in example 1 by a 100% sample adding and recycling method, taking 15mg of fasudil hydrochloride, precisely weighing and paralleling 8 parts, taking 2 parts of the fasudil hydrochloride, and determining the content of high piperazine in the sample; taking 6 parts of the piperazine solution, adding 0.2mL of the homopiperazine stock solution B, adding 5% of acetonitrile to the homopiperazine stock solution B to fix the volume, uniformly mixing the volume and the acetonitrile to obtain a solution with the standard recovery rate of 100%, and measuring the solution by the UPLC-MS method in example 1, wherein the results are shown in Table 4, and show that the homopiperazine recovery rate is 112-122% and the 6-pin recovery rate RSD is 4%, which indicates that the measuring method provided in example 1 has good repeatability.

TABLE 4 repeatability results

Example 5

Specificity test for the assay methods provided in example 1

5% acetonitrile is taken as a blank solvent, 5 mu L of each of the reference solution and the test solution in the example 1 is respectively injected into UPLC-MS, the UPLC-MS method in the example 1 is used for measuring, a chromatogram is recorded, and the result is shown in figure 3, wherein the reference numeral 1 in the figure is the blank solvent, the reference numeral 2 is the reference solution in the example 1, and the reference numeral 3 is the test solution in the example 1, and the result shows that the blank solvent and the test solution have no interference on the measurement of homopiperazine.

Example 6

Stability test of the assay methods provided in example 1

Taking the control solution in example 1, standing at room temperature, examining the stability of the solution, injecting samples according to the UPLC-MS method of example 1, recording the peak area of homopiperazine, calculating the peak area RSD of the control solution, and the result is shown in table 5, which shows that the peak area RSD of the control solution is 2% within 8h, thus indicating that the determination method provided in example 1 has good stability.

TABLE 5 control solution stability results

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

0.1% formic acid

Acetonitrile

1. A method for determining the content of homopiperazine in fasudil hydrochloride is characterized by comprising the following steps:

s3, detecting the test solution by using a UPLC-MS method to obtain the peak area of the test solution, and calculating the content of high piperazine in the test solution by using a response factor;

the chromatographic conditions of the UPLC-MS method in the S2 step and the S3 step are as follows: adopting a CSH C18 chromatographic column with the diameter of 2.1 multiplied by 100mm multiplied by 1.7 mu m and the column temperature is 35 ℃; the sample injection amount is 5 mu L; the detector is a mass spectrum detector, the ion source is ESI, the positive ion mode is adopted, and the ions are 101; taking 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate is 0.2 mL/min; the gradient elution procedure was: 0-1 min, wherein the volume percentage of the mobile phase B is 5%; 1-5 min, wherein the volume percentage of the mobile phase B is 5-90%; 5-5.1 min, wherein the volume percentage of the mobile phase B is from 90% to 5%; 5.1-7 min, and the volume percentage of the mobile phase B is 5%.