Detection method of lotinib

Abstract

The invention relates to the technical field, in particular to a detection method of lotirasodium, and solves the problem of lotirasodium content detection. The detection method comprises the following steps: a, preparing a lotinib reference substance solution and a lotinib sample solution; b, drawing a standard curve: respectively measuring 5-6 lotirasodium reference substance solutions with different concentrations by using HPLC (High Performance Liquid Chromatography), and carrying out linear regression by using the peak area of lotirasodium and the corresponding concentration value to obtain a standard curve and a linear regression equation of lotirasodium; and c, sample determination and result calculation: injecting the processed lotirazine sample solution into HPLC for determination, recording a chromatogram and a peak area, and calculating the content of lotirazine in the sample solution by using the linear regression equation obtained in the step b. The method is simple in sample pretreatment, simple and convenient to operate, accurate, efficient and easy to popularize, and can effectively monitor the content of lotirasodium in the synthesized sample and the conditions of related substances, so that the quality of a final product is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field, and in particular to a detection method of lotirana. Background Art

[0002] As an important drug ingredient, Lotiranab occupies an important position in the pharmaceutical field. As a key link in drug research and development and production, its purity and quality have a decisive impact on the efficacy and safety of the drug. In particular, accurate and reliable testing methods are crucial to ensure drug quality during drug production and quality control.

[0003] However, during the process development of Lotilan, we found that the existing detection technology could not fully meet the needs. The pharmacopoeia has not yet included the detection methods of Lotilan and related substances, which makes us lack unified and standardized detection methods when conducting drug quality control. The patents and literature that can be found at present are mainly focused on the process route or pharmacokinetics of Lotilan, and there are few reports on its specific detection methods.

[0004] Specifically, the existing detection methods face prominent problems such as complex operation, low accuracy and poor reproducibility in the detection of lotirana and its related substances. These problems directly lead to the reduction of drug quality detection efficiency, the increase of operation difficulty, and the difficulty in meeting the needs of high-precision and large-scale production quality control, which increases the risk of drug quality. Therefore, in view of the many deficiencies in the existing technology, we urgently need an innovative lotirana detection method to solve these problems. Summary of the invention

[0005] The purpose of the present invention is to provide a detection method for lotirana, which solves the problem that the detection method for lotirana and related substances has not been included in the pharmacopoeia in the prior art, which makes us lack a unified and standardized detection method when performing drug quality control.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for detecting lotirana comprises the following steps:

[0008] Step 1: preparing a solution, accurately weighing an appropriate amount of a lotirana reference substance, dissolving and diluting it to a desired concentration with a suitable solvent to prepare a lotirana reference substance solution, preparing a lotirana synthetic sample according to a synthetic process, and obtaining a lotirana synthetic sample solution to be tested after appropriate treatment;

[0009] Step 2: Draw a standard curve, use an HPLC (high performance liquid chromatography) instrument, set appropriate chromatographic conditions, such as mobile phase, flow rate, column temperature, and detection wavelength, and measure different concentrations of lotirana reference solutions respectively to ensure that each concentration point has a good chromatographic peak shape and separation, record the lotirana peak area corresponding to each concentration point, use the peak area as the ordinate and the corresponding concentration value as the abscissa, perform linear regression analysis, and obtain the standard curve and linear regression equation of lotirana through linear regression analysis;

[0010] Step 3: Sample determination and result calculation, the synthetic sample is subjected to necessary treatment, such as dissolution, purification, concentration, etc., to obtain a lotirana sample solution suitable for HPLC determination, the treated lotirana sample solution is injected into the HPLC instrument for determination, and the chromatogram and peak area are recorded;

[0011] Step 4: Determine the presence and purity of lotirana in the sample solution based on the peak shape, separation, peak area and other information of the chromatogram. Use the linear regression equation obtained in step 2 to substitute the peak area of ​​the sample solution into the equation to calculate the content of lotirana in the sample solution. Perform necessary verification and check on the calculation results.

[0012] Preferably, the preparation method of the lotirana reference solution is as follows: first, accurately weigh an appropriate amount of lotirana reference, then dissolve it in acetonitrile / water solution as a solvent, and dilute it to an initial concentration of 0.5-1.0 mg / mL. In order to ensure the accuracy and reliability of the standard curve, it is necessary to dilute this initial solution step by step to obtain a series of lotirana reference solutions of different concentrations for subsequent HPLC determination and linear regression analysis.

[0013] Preferably, the preparation method of the lotirana sample solution is as follows: first, take an appropriate amount of the synthesized lotirana sample, then dissolve it in acetonitrile / water solution as a solvent, and dilute it to a certain volume. In order to remove impurities and particles in the solution, the solution needs to be filtered to finally obtain a clear and transparent lotirana sample solution for HPLC determination.

[0014] Preferably, the specific ratio of acetonitrile / water solution is acetonitrile: water (70:30). This ratio is optimized based on the solubility of lotirana and the separation effect of HPLC, which can ensure the good dissolution of lotirana in the solution and obtain good chromatographic separation and peak shape in HPLC determination.

[0015] Preferably, the chromatographic conditions for HPLC determination are as follows: chromatographic column: C 184.6*250mm, 5μm; column temperature: 30±5℃; wavelength: 205±2nm; flow rate: 0.8~1.2mL / min; injection volume: 5~10μL; running time: 45~55min; mobile phase A: acetonitrile; mobile phase B: 0.1% trifluoroacetic acid aqueous solution.

[0016] Preferably, the gradient elution program: the gradient elution program starts from 0 minutes, the ratio of mobile phase A and B is 70% and 30%, which is maintained unchanged for 30 minutes, and then at 30.5 minutes, the ratio of mobile phase A is rapidly increased to 90%, and the ratio of mobile phase B is correspondingly reduced to 10%, and maintained for 40 minutes, and then at 40.5 minutes, the ratio of mobile phase A and B is rapidly adjusted back to 70% and 30%, and maintained until the end of 50 minutes.

[0017] Preferably, the volume concentration of the trifluoroacetic acid aqueous solution in the mobile phase is 0.1%.

[0018] The present invention has at least the following beneficial effects:

[0019] The practical application effect of the technical solution is demonstrated by specific examples. As in Example 1, the retention time of lotirana is 11.265min, the content of lotirana in the sample is 89.5%, and the retention time of the main peak of the chromatogram of the reference solution and the sample solution is consistent, which illustrates the accuracy and reliability of the determination method. Example 2 and Example 3 also respectively show the determination results under different chromatographic conditions and sample amounts, further verifying the applicability and stability of the technical solution. The present invention provides a detailed method for the determination of lotirana content by accurately preparing reference substances and sample solutions, drawing standard curves, optimizing HPLC chromatographic conditions and gradient elution procedures, etc., with the beneficial effects of high accuracy, good repeatability, strong selectivity and high sensitivity, and provides strong technical support for the quality control and research of lotirana. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0021] Figure 1 is a chromatogram of the reference substance solution of Example 1 of the present invention;

[0022] Figure 2 is a chromatogram of the sample solution of Example 1 of the present invention;

[0023] Figure 3 is a chromatogram of the reference substance solution of Example 2 of the present invention;

[0024] Figure 4 is a chromatogram of the sample solution of Example 2 of the present invention;

[0025] Figure 5 is a chromatogram of the reference substance solution of Example 3 of the present invention;

[0026] Figure 6 is a chromatogram of the sample solution of Example 3 of the present invention;

[0027] Figure 7 It is a standard curve diagram of Example 1 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] Reference Figure 1-7 , including the following steps:

[0030] Step 1: preparing a solution, accurately weighing an appropriate amount of a lotirana reference substance, dissolving and diluting it to a desired concentration with a suitable solvent to prepare a lotirana reference substance solution, preparing a lotirana synthetic sample according to a synthetic process, and obtaining a lotirana synthetic sample solution to be tested after appropriate treatment;

[0031] Step 2: Draw a standard curve, use an HPLC (high performance liquid chromatography) instrument, set appropriate chromatographic conditions, such as mobile phase, flow rate, column temperature, and detection wavelength, and measure different concentrations of lotirana reference solutions respectively to ensure that each concentration point has a good chromatographic peak shape and separation, record the lotirana peak area corresponding to each concentration point, use the peak area as the ordinate and the corresponding concentration value as the abscissa, perform linear regression analysis, and obtain the standard curve and linear regression equation of lotirana through linear regression analysis;

[0032] Step 3: Sample determination and result calculation, the synthetic sample is subjected to necessary treatment, such as dissolution, purification, concentration, etc., to obtain a lotirana sample solution suitable for HPLC determination, the treated lotirana sample solution is injected into the HPLC instrument for determination, and the chromatogram and peak area are recorded;

[0033] Step 4: Determine the presence and purity of lotirana in the sample solution based on the peak shape, separation, peak area and other information of the chromatogram. Use the linear regression equation obtained in step 2 to substitute the peak area of ​​the sample solution into the equation to calculate the content of lotirana in the sample solution. Perform necessary verification and check on the calculation results.

[0034] Further

[0035] Reference Figure 1-7 The preparation method of the lotirana reference solution is specifically as follows: first, accurately weigh an appropriate amount of lotirana reference, then dissolve it in acetonitrile / water solution as a solvent, and dilute it to an initial concentration of 0.5-1.0 mg / mL. In order to ensure the accuracy and reliability of the standard curve, it is necessary to dilute this initial solution step by step to obtain a series of lotirana reference solutions of different concentrations for subsequent HPLC determination and linear regression analysis.

[0036] Further

[0037] Reference Figure 1-7 The preparation method of the lotirana sample solution is specifically as follows: first, take an appropriate amount of the synthesized lotirana sample, then dissolve it in acetonitrile / water solution as a solvent, and dilute it to a certain volume. In order to remove impurities and particles in the solution, the solution needs to be filtered to finally obtain a clear and transparent lotirana sample solution for HPLC determination.

[0038] Further

[0039] Reference Figure 1-7 The specific ratio of the acetonitrile / water solution is acetonitrile: water (70:30). This ratio is optimized based on the solubility of lotirana and the separation effect of HPLC, which can ensure the good dissolution of lotirana in the solution and obtain good chromatographic separation and peak shape during HPLC determination.

[0040] Further

[0041] Reference Figure 1-7 The chromatographic conditions for the HPLC determination are as follows: chromatographic column: C184.6*250mm, 5μm; column temperature: 30±5°C; wavelength: 205±2nm; flow rate: 0.8-1.2mL / min; injection volume: 5-10μL; running time: 45-55min; mobile phase A: acetonitrile; mobile phase B: 0.1% trifluoroacetic acid aqueous solution.

[0042] Further

[0043] Reference Figure 1-7 , the gradient elution program: the gradient elution program starts from 0 minute, the ratio of mobile phase A and B is 70% and 30%, which is kept unchanged for 30 minutes, and then at 30.5 minutes, the ratio of mobile phase A is rapidly increased to 90%, and the ratio of mobile phase B is correspondingly reduced to 10%, and is kept until 40 minutes, and then at 40.5 minutes, the ratio of mobile phase A and B is rapidly adjusted back to 70% and 30%, and is kept until the end of 50 minutes, and the volume concentration of trifluoroacetic acid aqueous solution in the mobile phase is 0.1%.

[0044] The sources of materials in the following methods are as follows: synthetic sample (homemade); lotirana (CAS No.: 1369852-71-0, Medisin Biopharmaceutical Technology Chengdu Co., Ltd.); acetonitrile (CAS No.: 75-05-8, Merck); trifluoroacetic acid (CAS No.: 76-05-1, Aladdin).

[0045] In summary:

[0046] Embodiment 1:

[0047] Chromatographic conditions

[0048] Chromatographic column: C 18 4.6*250mm, 5μm;

[0049] Column temperature: 30±5℃;

[0050] Wavelength: 205nm;

[0051] Flow rate: 1.0 mL / min;

[0052] Injection volume: 5 μL;

[0053] Running time: 50min;

[0054] Mobile phase A: acetonitrile;

[0055] Mobile phase B: 0.1% trifluoroacetic acid in water;

[0056] Gradient elution program:

[0057] Time min Mobile phase A% Mobile phase B% 0 70 30 30 70 30 30.5 90 10 40 90 10 40.5 70 30 50 70 30

[0058] a. Preparation of lotirana reference solution: accurately weigh 100 mg of lotirana reference substance, dissolve and dilute to a mass concentration of 1.0 mg / mL with acetonitrile / water solution (70:30), shake well, and use as reference solution 1; dilute reference solution 1 stepwise with acetonitrile / water solution (70:30) to obtain reference solution 2, reference solution 3, reference solution 4, and reference solution 5;

[0059] b. Prepare the lotirana sample solution according to the above operation, accurately weigh 10 mg of the synthetic sample, dissolve it in acetonitrile / water solution (70:30) to make up the volume, and filter the supernatant through a microporous filter membrane to obtain the lotirana sample solution;

[0060] c. Draw the standard curve: Turn on the liquid chromatograph, wait until the instrument parameters meet the requirements, balance the baseline for 40 minutes, inject 5 reference solutions in sequence, and record the chromatogram and peak area. Perform linear regression with the peak area of ​​lotirana and the corresponding concentration value to obtain the standard curve and linear regression equation of lotirana;

[0061] In this example, the retention time of lotirana is 11.265min. The contents of the reference solution are calculated as x1=25.13, x2=125.6, x3=251.3, x4=502.6, and x5=1005.2, respectively. The corresponding injection peak areas are y1=648859, y2=3227658, y3=5855236, y4=11374193, and y5=21717196, respectively. The peak area of ​​lotirana and the corresponding content are linearly regressed to obtain the linear regression equation:

[0062] y=21510x+293189

[0063] Wherein, x: the content of lotirana; y: the peak area of ​​the sample;

[0064] d. Sample determination and result calculation: Inject the sample solution into HPLC for determination, record the chromatogram and peak area, and use the linear regression equation obtained in step c to calculate the content of lotirana in the sample solution. The peak area of ​​the sample is calculated as yi = 19546810, which is substituted into the univariate linear regression equation obtained in step c to calculate the content of lotirana in the chromatogram of the sample solution as xi = 895. Then the content of lotirana in the sample is: 89.5%, as shown in Figure 1-2 As shown, the main peak retention time of the chromatogram of the reference solution of Example 1 is 11.468 min, and the retention time of the sample solution is 11.479 min.

[0065] Embodiment 2:

[0066] Chromatographic conditions

[0067] Chromatographic column: C 18 4.6*250mm, 5μm;

[0068] Column temperature: 30±5℃;

[0069] Wavelength: 205nm;

[0070] Flow rate: 1.2 mL / min;

[0071] Injection volume: 5 μL;

[0072] Running time: 50min;

[0073] Mobile phase A: acetonitrile;

[0074] Mobile phase B: 0.1% trifluoroacetic acid in water;

[0075] Gradient elution program:

[0076] Time min Mobile phase A% Mobile phase B% 0 70 30 30 70 30 30.5 90 10 40 90 10 40.5 70 30 50 70 30

[0077] a. Preparation of lotirana reference solution: accurately weigh 50 mg of lotirana reference substance, dissolve and dilute to a mass concentration of 0.5 mg / mL with acetonitrile / water solution (70:30), shake well, and use as reference solution 1; dilute reference solution 1 stepwise with acetonitrile / water solution (70:30) to obtain reference solution 2, reference solution 3, reference solution 4, and reference solution 5;

[0078] b. Prepare the lotirana sample solution according to the above operation, accurately weigh 5 mg of the synthetic sample, dissolve it in acetonitrile / water solution (70:30) to make up the volume, and filter the supernatant with a microporous filter membrane to obtain the lotirana sample solution;

[0079] c. Draw the standard curve: Turn on the liquid chromatograph, wait until the instrument parameters meet the requirements, balance the baseline for 40 minutes, inject 5 reference solutions in sequence, and record the chromatogram and peak area. Perform linear regression with the peak area of ​​lotirana and the corresponding concentration value to obtain the standard curve and linear regression equation of lotirana;

[0080] d. Sample determination and result calculation: Inject the sample solution into HPLC for determination, record the chromatogram and peak area, and use the linear regression equation obtained in step c to calculate the content of lotirana in the sample solution. Figure 3-4 As shown, the main peak retention time of the chromatogram of the reference solution of Example 2 is 14.136 min, and the retention time of the sample solution is 14.107 min.

[0081] Embodiment three:

[0082] Chromatographic conditions

[0083] Chromatographic column: C 18 4.6*250mm, 5μm;

[0084] Column temperature: 30±5℃;

[0085] Wavelength: 207nm;

[0086] Flow rate: 1.0 mL / min;

[0087] Injection volume: 5 μL;

[0088] Running time: 50min;

[0089] Mobile phase A: acetonitrile;

[0090] Mobile phase B: 0.1% trifluoroacetic acid in water;

[0091] Gradient elution program:

[0092]

[0093]

[0094] a. Preparation of lotirana reference solution: accurately weigh 100 mg of lotirana reference substance, dissolve and dilute to a mass concentration of 1.0 mg / mL with acetonitrile / water solution (70:30), shake well, and use as reference solution 1; dilute reference solution 1 stepwise with acetonitrile / water solution (70:30) to obtain reference solution 2, reference solution 3, reference solution 4, and reference solution 5;

[0095] b. Prepare the lotirana sample solution according to the above operation, accurately weigh 10 mg of the synthetic sample, dissolve it in acetonitrile / water solution (70:30) to make up the volume, and filter the supernatant through a microporous filter membrane to obtain the lotirana sample solution;

[0096] c. Draw the standard curve: Turn on the liquid chromatograph, wait until the instrument parameters meet the requirements, balance the baseline for 40 minutes, inject 5 reference solutions in sequence, and record the chromatogram and peak area. Perform linear regression with the peak area of ​​lotirana and the corresponding concentration value to obtain the standard curve and linear regression equation of lotirana;

[0097] d. Sample determination and result calculation: Inject the sample solution into HPLC for determination, record the chromatogram and peak area, and calculate the content of lotirana in the sample solution using the linear regression equation obtained in step c.

[0098] like Figure 5-6 As shown, the main peak retention time of the chromatogram of the reference solution of Example 3 is 11.282 min, and the retention time of the sample solution is 11.278 min.

[0099] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for detecting lotirana, characterized in that: It includes the following steps: Step 1: preparing a solution, accurately weighing an appropriate amount of a lotirana reference substance, dissolving and diluting it to a desired concentration with a suitable solvent to prepare a lotirana reference substance solution, preparing a lotirana synthetic sample according to a synthetic process, and obtaining a lotirana synthetic sample solution to be tested after appropriate treatment; Step 2: Draw a standard curve, use an HPLC (high performance liquid chromatography) instrument, set appropriate chromatographic conditions, such as mobile phase, flow rate, column temperature, and detection wavelength, and measure different concentrations of lotirana reference solutions respectively to ensure that each concentration point has a good chromatographic peak shape and separation, record the lotirana peak area corresponding to each concentration point, use the peak area as the ordinate and the corresponding concentration value as the abscissa, perform linear regression analysis, and obtain the standard curve and linear regression equation of lotirana through linear regression analysis; Step 3: Sample determination and result calculation, perform necessary treatment on the synthetic sample, such as dissolution, purification, concentration, etc., to obtain a lotirana sample solution suitable for HPLC determination, inject the treated lotirana sample solution into the HPLC instrument for determination, and record the chromatogram and peak area; Step 4: Determine the presence and purity of lotirana in the sample solution based on the peak shape, separation, peak area and other information of the chromatogram. Use the linear regression equation obtained in step 2 to substitute the peak area of ​​the sample solution into the equation to calculate the content of lotirana in the sample solution. Perform necessary verification and check on the calculation results.

2. The method for detecting lotirana according to claim 1, characterized in that: The preparation method of the lotirana reference solution is specifically as follows: first, accurately weigh an appropriate amount of lotirana reference, then dissolve it in acetonitrile / water solution as a solvent, and dilute it to an initial concentration of 0.5-1.0 mg / L. In order to ensure the accuracy and reliability of the standard curve, it is necessary to dilute this initial solution step by step to obtain a series of lotirana reference solutions of different concentrations for subsequent HPLC determination and linear regression analysis.

3. The method for detecting lotirana according to claim 1, characterized in that: The preparation method of the lotirana sample solution is specifically as follows: first, take an appropriate amount of the synthesized lotirana sample, then dissolve it in acetonitrile / water solution as a solvent, and dilute it to a certain volume. In order to remove impurities and particles in the solution, the solution needs to be filtered to finally obtain a clear and transparent lotirana sample solution for HPLC determination.

4. The method for detecting lotirana according to claim 3, characterized in that: The specific ratio of the acetonitrile / water solution is acetonitrile: water (70:30). This ratio is optimized based on the solubility of lotirana and the separation effect of HPLC, which can ensure the good dissolution of lotirana in the solution and obtain good chromatographic separation and peak shape during HPLC determination.

5. The method for detecting lotirana according to claim 1, characterized in that: The chromatographic conditions for the HPLC determination are as follows: chromatographic column: C 18 4.6*250mm, 5μm; column temperature: 30±5℃; Wavelength: 205±2nm; flow rate: 0.8~1.2mL / min; injection volume: 5~10μL; running time: 45~55min; mobile phase A: acetonitrile; mobile phase B: 0.1% trifluoroacetic acid aqueous solution.

6. The method for detecting lotirana according to claim 1, characterized in that: The gradient elution program: The gradient elution program starts from 0 minutes, the ratio of mobile phase A and B is 70% and 30%, which is kept unchanged for 30 minutes, and then at 30.5 minutes, the ratio of mobile phase A is rapidly increased to 90%, and the ratio of mobile phase B is correspondingly reduced to 10%, and maintained for 40 minutes, and then at 40.5 minutes, the ratio of mobile phase A and B is rapidly adjusted back to 70% and 30%, and maintained until the end of 50 minutes.

7. The method for detecting lotirana according to claim 6, characterized in that: The volume concentration of the trifluoroacetic acid aqueous solution in the mobile phase is 0.1%.

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