A high performance liquid chromatography analysis method for detecting glapiron content

Abstract

The invention discloses a method for detecting the content of grapipiron. The invention adopts a reverse phase high performance liquid chromatograph to measure the standard product and the test product of grapipiron, and obtains the content of the sample to be tested according to the standard curve. The method can accurately measure the content of grapipiron, and the detection method without interference is reliable and stable, with high sensitivity and high theoretical plate number. At the same time, the operation is relatively simple, and the content of grapipiron can be effectively measured, which is conducive to ensuring the quality of grapipiron raw materials and preparations.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a reversed-phase high performance liquid chromatography analysis method for detecting glapiron content. Background Art

[0002] Glapiron is a prostaglandin E2 subtype 4 (E4) receptor antagonist that can be used to treat pain and inflammation. Glapiron formulations are valuable because they can alter the pharmacokinetic properties of drugs in vivo, such as peak plasma concentration, time to peak concentration, half-life, and area under the plasma concentration curve (Cmax, Tmax, t1 / 2, and AUC, respectively).

[0003] At present, there is no patent literature report on the method for detecting the grapipiron content in grapipiron preparations. Therefore, developing a method for detecting the grapipiron content in grapipiron preparations with high sensitivity, reliability, stability and good separation is extremely important for achieving quality control of grapipiron preparations. Summary of the invention

[0004] The object of the present invention is to overcome at least one disadvantage of the prior art and provide a high performance liquid chromatography analysis method for detecting glapilon content.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a high performance liquid chromatography analysis method for detecting glapilon content, comprising the following steps:

[0007] 1) preparing a standard solution containing a glapiron standard;

[0008] 2) preparing a test sample solution in which the test sample of glapiron is dissolved;

[0009] 3) Using reversed-phase high performance liquid chromatography to measure the chromatograms of the standard solution and the sample solution to be tested, and calculating the content of the sample to be tested based on the chromatograms.

[0010] In some examples, it is characterized in that the detection conditions of the reversed-phase high performance liquid chromatography are: the mobile phase consists of mobile phase A and mobile phase B, wherein acetonitrile is mobile phase A, trifluoroacetic acid solution is mobile phase B, and the mobile phase is used for gradient elution.

[0011] In some examples, the mass concentration of trifluoroacetic acid in the trifluoroacetic acid solution is 0.05% to 0.15%.

[0012] In some examples, the flow rate of the mobile phase in the reverse phase HPLC is 0.6-1 mL / min.

[0013] In some examples, the order of gradient elution time and volume concentration of acetonitrile in the mobile phase in the reversed-phase high performance liquid chromatography is:

[0014] 1) 0-15min, 60-70% volume concentration operation;

[0015] 2) 15-25 min, 10-20% volume concentration operation;

[0016] 3) 25 to 35 minutes, operation at 60 to 70% volume concentration.

[0017] In some examples, the concentration of glapiron in the standard solution is 0.3-1 mg / mL.

[0018] In some examples, the solvent of the standard solution and the test solution is acetonitrile aqueous solution with a volume concentration of 40% to 60%.

[0019] In some examples, the injection volume of the HPLC is 10 μL to 20 μL.

[0020] In some examples, the HPLC column temperature is 20°C to 35°C.

[0021] In some examples, the detection wavelength of the HPLC is 210 nm to 230 nm.

[0022] The beneficial effects of the present invention are as follows: the high performance liquid phase analysis method for detecting the content of grapipiron provided by the present invention has high detection sensitivity, high stability, high reliability, high theoretical plate number, and relatively simple operation, and can effectively measure the content of grapipiron, which is beneficial to ensuring the quality of grapipiron raw materials and preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the chromatogram of A glapiron standard solution;

[0024] Figure 2 is the chromatogram of B glapiron standard solution;

[0025] Figure 3 is the chromatogram of C glapiron standard solution;

[0026] Figure 4 This is the chromatogram of the glapiron test solution;

[0027] Figure 5 This is the chromatogram of the glapirone standard solution in the system suitability test for the determination of glapirone content;

[0028] Figure 6 It is the linear relationship diagram of grapillon;

[0029] Figure 7 It is the chromatogram of the glapiron test solution in the comparative example. DETAILED DESCRIPTION

[0030] The following disclosure provides many different implementations or examples for implementing different solutions.

[0031] In the following examples, comparative examples and reagents used in the tests, acetonitrile was imported from abroad and of HPLC grade, brand Fisher, water was purified water to reduce baseline noise, and the high performance liquid chromatograph used was a Waters Arc high performance liquid chromatograph.

[0032] In the following examples, comparative examples and tests, unless otherwise specified, the chromatographic measurement conditions are as follows:

[0033] Chromatographic column: Agilent ZORBAX Eclipse Plus C18, size 4.6×250mm, 5μm;

[0034] Mobile phase: A: acetonitrile, B: 0.1% trifluoroacetic acid solution;

[0035] Flow rate: 1 mL / min;

[0036] Column temperature: 30°C;

[0037] Injection volume: 10 μL;

[0038] Detection wavelength: 230nm;

[0039] Gradient elution program:

[0040] When the time is 0.0min, the volume fraction of A in the mobile phase is 70%, and the volume fraction of B is 30%;

[0041] When the time is 15.0 min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;

[0042] When the time is 25.0 min, the volume fraction of A in the mobile phase is 70%, and the volume fraction of B is 30%;

[0043] When the time is 35.0 min, the volume fraction of A in the mobile phase is 70%, and the volume fraction of B is 30%.

[0044] Reverse Phase High Performance Liquid Chromatographic Analysis Method for Detecting Glapiron Content

[0045] Preparation of standard solution: Weigh approximately 12.8 mg, 25.0 mg and 40.1 mg of grapipiron respectively, accurately weigh in a 50 mL volumetric flask, add appropriate volume of 60% acetonitrile aqueous solution to dissolve and dilute to scale, mix well, and the grapipiron concentrations are 0.256, 0.50, and 0.802 mg / mL, corresponding to the standard solutions numbered A to C respectively.

[0046] The detection chromatogram of standard solution A is as follows Figure 1 As shown. Figure 1 It can be seen that the retention time of glapilon is 6.526 min, the peak shape is good, and the peak purity is high (≥99.0%).

[0047] The detection chromatogram of B standard solution is as follows Figure 2 As shown. Figure 2 It can be seen that the retention time of glapilon is 6.477 min, the peak shape is good, and the peak purity is high (≥99.0%).

[0048] The detection chromatogram of C standard solution is as follows Figure 3 As shown. Figure 3 It can be seen that the retention time of glapilon is 6.422 min, the peak shape is good, and the peak purity is high (≥99.0%).

[0049] Combination Figures 1 to 3 It can be seen from the liquid phase spectrum that the standard curve of grapipiron measured under the chromatographic conditions of the present invention has a wide range of applicability and can accurately measure both high and low contents.

[0050] Detection of glapilon test solution:

[0051] Preparation of test solution: Weigh about 25.35 mg of grapipiron raw material sample, accurately weigh it in a 50 mL volumetric flask, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the scale, mix well, and the grapipiron concentration is 0.507 mg / mL.

[0052] The test chromatogram of the test solution is as follows Figure 4 As shown, from Figure 4 It can be seen that the retention time of glapiron is 6.489 min, the peak shape is good, the peak purity is high (≥98.0%), and the determined glapiron is not interfered by other components of the drug sample to be tested.

[0053] System suitability test for determination of glapiron content

[0054] Preparation of standard solution: Weigh 25.1 mg of grapipiron and accurately weigh it in a 50 mL volumetric flask. Add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark. Mix well to obtain a grapipiron concentration of 0.502 mg / mL. Take the standard solution and repeat the injection 6 times. The test results are shown in Table 1 and Figure 5 As shown:

[0055] Table 1. System suitability test results

[0056]

[0057] As can be seen from Table 1, the same grapipirone sample solution was injected 6 times, and the RSD of the grapipirone peak area was 0.13%, which met the requirement of RSD≤2.0%, indicating that the method of the present invention has good system adaptability and high test credibility.

[0058] Example

[0059] Preparation of standard solution: according to the sample concentration of 50%, 80%, 100%, 120% and 150%, weigh 20-100 mg of grapipiron standard into a 20 ml volumetric flask, dilute to the scale with solvent, shake well, as the stock solution, accurately measure 0.5 ml, 0.8 ml, 1.0 ml, 1.2 ml, 1.5 ml into a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain grapipiron standard concentrations of 0.25 mg / ml, 0.40 mg / ml, 0.50 mg / ml, 0.60 mg / ml, 0.75 mg / ml, corresponding to standard solutions numbered 1 to 5, respectively.

[0060] Take standard solutions of different mass concentrations No. 1 to 5 and inject them respectively, and measure the peak area and standard concentration of each standard solution as shown in Table 2:

[0061] Table 2. Results of linear relationship investigation

[0062] serial number Concentration (mg / ml) Peak area 1 0.25 4102632 2 0.40 6583446 3 0.50 8279852 4 0.60 9865443 5 0.75 12405846

[0063] From Table 2 and Figure 6 It can be seen that the peak area of ​​​​Glapiron shows a good linear relationship with the concentration, and the standard linear R 2 =0.9998, in line with the pharmacopoeia R 2 ≥0.99, which indicates that the detection method has good linearity in the concentration range of 0.25-0.75 mg / ml.

[0064] Preparation of test samples: Weigh 6 portions of the same glapiron bulk drug sample: (25.11 mg, 25.21 mg, 25.15 mg, 25.30 mg, 25.17 mg, 25.22 mg), accurately weigh each into a 50 mL volumetric flask, dissolve and dilute to the scale with 60% acetonitrile aqueous solution by volume, mix well, and obtain glapiron solutions of different mass concentrations, which correspond to standard solutions No. 1 to 6: 0.502 mg / mL, 0.504 mg / mL, 0.503 mg / mL, 0.506 mg / mL, 0.503 mg / mL, 0.504 mg / mL.

[0065] Take samples of the test solution No. 6 to No. 11 with different mass concentrations and inject them separately, and then Figure 6 The content of each test solution was calculated using the standard curve equation in Table 3:

[0066] Table 3. Repeatability test results

[0067]

[0068] As can be seen from Table 3, the average content of grapipiron in the test sample is 98.29%, and the RSD% of grapipiron content is 0.26%, which meets the requirement that the content is between 98.0% and 102.0%, and the RSD% is not greater than 2.0%, indicating that the method of the present invention has good reproducibility.

[0069] Comparative Example

[0070] To prepare the test solution, weigh about 25.29 mg of grapipirone and accurately weigh it in a 50 mL volumetric flask. Add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the scale. Mix well to obtain a grapipirone concentration of 0.506 mg / mL.

[0071] Chromatographic column: Agilent ZORBAX Eclipse Plus C18, size 4.6×250mm, 5μm;

[0072] Mobile phase: A: acetonitrile, B: 0.1% trifluoroacetic acid solution;

[0073] Flow rate: 1.2 mL / min;

[0074] Column temperature: 30°C;

[0075] Injection volume: 10 μL;

[0076] Detection wavelength: 230nm;

[0077] Gradient elution program:

[0078] When the time is 0.0min, the volume fraction of A in the mobile phase is 70%, and the volume fraction of B is 30%;

[0079] When the time is 15.0 min, the volume fraction of A in the mobile phase is 20%, and the volume fraction of B is 80%;

[0080] When the time is 25.0 min, the volume fraction of A in the mobile phase is 70%, and the volume fraction of B is 30%;

[0081] When the time is 35.0 min, the volume fraction of A in the mobile phase is 70%, and the volume fraction of B is 30%;

[0082] The test results of the comparative example are: Figure 7 As shown, the impurities cannot be completely separated from each other, which also affects the separation of the main peak, and the content of glapilon cannot be accurately detected.

[0083] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A high performance liquid chromatography analysis method for detecting the content of glapiron, characterized in that: The following steps are involved: 1) Prepare a standard solution containing glapiron standard; 2) preparing a test sample solution in which a test sample of glapiron is dissolved, wherein the test sample solution includes a glapiron bulk drug sample, and then adding an acetonitrile aqueous solution having a volume concentration of 60% to dissolve the sample; 3) using reverse phase high performance liquid chromatography to measure the chromatograms of the standard solution and the sample solution to be tested, and calculating the content of the sample to be tested based on the chromatograms; The detection conditions of the reversed-phase high performance liquid chromatography are as follows: the mobile phase consists of a mobile phase A and a mobile phase B, wherein acetonitrile is the mobile phase A, a trifluoroacetic acid solution with a mass concentration of 0.05% to 0.15% is the mobile phase B, the mobile phase is used for gradient elution at a flow rate of 0.6 to 1 mL / min, the detection wavelength of the high performance liquid chromatography is 210 nm to 230 nm, the chromatographic column of the high performance liquid chromatography is Agilent ZORBAX Eclipse Plus C18, and the gradient elution time in the reversed-phase high performance liquid chromatography and the volume concentration of acetonitrile in the mobile phase are in the following order: 1) 0-15min, 60-70% volume concentration operation; 2) 15-25min, 10-20% volume concentration operation; 3) 25 to 35 minutes, run at 60 to 70% volume concentration.

2. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The concentration of glapiron in the standard solution is 0.3-1 mg / mL.

3. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The solvent of the standard solution and the test solution is acetonitrile aqueous solution with a volume concentration of 40% to 60%.

4. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The injection volume of the high performance liquid chromatography is 10 μL to 20 μL.

5. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The chromatographic column temperature of the high performance liquid chromatography is 20°C to 35°C.

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