CN108020603B

CN108020603B - High performance liquid phase analysis method of kebomei

Info

Publication number: CN108020603B
Application number: CN201610952274.XA
Authority: CN
Inventors: 苗瑞娜; 刘跃跃; 林彬
Original assignee: Jiangsu Wanbang Biopharmaceutical Group Co ltd
Current assignee: Jiangsu Wanbang Biopharmaceutical Group Co ltd
Priority date: 2016-11-02
Filing date: 2016-11-02
Publication date: 2020-09-08
Anticipated expiration: 2036-11-02
Also published as: CN108020603A

Abstract

The invention discloses a high performance liquid analysis method of Colume, which comprises the following steps: 1) dissolving and diluting the cobimel with acetonitrile into a solution containing 0.2-2 mg of the cobimel in each 1mL of the solution to be used as a test solution; 2) adopting a chromatographic column of octadecyl bonded silica gel, preparing an ultraviolet detector, detecting the wavelength of 200-280 nm, and eluting by taking acetonitrile-phosphoric acid solution or acetonitrile-phosphoric acid solution as a mobile phase with the flow rate of 0.5-3.0 mL/min; 3) injecting the sample solution obtained in the step 1) into a high performance liquid chromatograph, wherein the sample injection volume is 1-100 mu L, and recording a chromatogram. The method has the advantages of simple process, high precision, good stability, and good reproducibility.

Description

High performance liquid phase analysis method of kebomei

Technical Field

The invention relates to a high performance liquid phase analysis method of Colume.

Background

Cobume (Roxadustat) is a small molecule inhibitor developed by fibraurea (fibrodin) corporation of america for regulating the stability or activity of the hypoxia inducible factor HIF, an intentional transcription factor that is very sensitive to cellular hypoxia, and plays a key role in the homeostasis of oxygen in the organism.

Bomei is used as a novel oral hypoxia inducible factor prolyl hydroxylase inhibitor (HIF-PHIs), and has good effect on treating anemia patients suffering from nephropathy. These drugs stimulate the body to produce an effect similar to that in the plateau area, and promote the body to produce more red blood cells for transporting oxygen required by the body, thereby improving the symptoms of the end-stage renal disease and anemia in chronic renal patients accompanied by a decrease in red blood cells (carrying oxygen in the body) and a decrease in hemoglobin (the protein required by red blood cells to carry oxygen); in addition, compared with the current methods for treating anemia, the method comprises the recombinant human erythropoietin, erythropoiesis stimulating medicines, intravenous injection iron agents and other medicines, and has higher safety.

The cabomeet has an isoquinoline compound structure, the chemical name is 2- (4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-formamido) acetic acid, and the molecular formula is as follows: C19H16N2O5, CAS number 808118-40-3, structural formula as follows:

in order to ensure the subsequent development and production quality of the Colubmei, the purity of the Colubmei is required to be detected. Therefore, it is very urgent for pharmaceutical manufacturers to research and obtain a detection method for checking and measuring the content of substances related to kebotai.

Disclosure of Invention

The invention provides a high performance liquid phase analysis method of Bomei, which is simple, high in precision, good in stability and good in reproducibility.

In order to solve the above problems, the present invention adopts a technical solution that a high performance liquid chromatography method of kebotai comprises the steps of:

1) dissolving and diluting the cobimel with acetonitrile into a solution containing 0.2-2 mg of the cobimel in each 1mL of the solution to be used as a test solution;

2) adopting a chromatographic column of octadecyl bonded silica gel, preparing an ultraviolet detector, detecting the wavelength of 200-280 nm, and eluting by taking acetonitrile-phosphoric acid solution or acetonitrile-phosphoric acid solution as a mobile phase with the flow rate of 0.5-3.0 mL/min;

3) injecting the sample solution obtained in the step 1) into a high performance liquid chromatograph, wherein the sample injection volume is 1-100 mu L, and recording a chromatogram.

Wherein, the volume content of acetonitrile in the mobile phase is 10-90%, preferably 40-80%; the volume content of the phosphoric acid solution or the phosphate solution is 10-90 percent, and the optimal volume content is 20-60 percent.

When the mobile phase is eluted, gradient elution is preferably adopted, and the procedure of the gradient elution is as follows: changing the volume content of the phosphoric acid solution or the phosphate solution from 60% to 20% and the volume content of the acetonitrile from 40% to 80% for 0-20 min; 20-25min, keeping the volume content of the phosphoric acid solution or the phosphate solution at 20%, and keeping the volume content of the acetonitrile at 80%; changing the volume content of the phosphoric acid solution or the phosphate solution from 20% to 60% and the volume content of the acetonitrile from 80% to 40% within 25-27 min; 27-35min, the volume content of the phosphoric acid solution or the phosphate solution is kept at 60%, and the volume content of the acetonitrile is kept at 40%.

On the basis of the technical scheme, the volume concentration of phosphoric acid in the phosphoric acid solution is 0.01-10%, and the optimal volume concentration is 0.1%;

on the basis of the technical scheme, the phosphate solution is preferably a mixed solution of one or more of potassium dihydrogen phosphate solution, sodium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, disodium hydrogen phosphate solution, potassium phosphate solution and sodium phosphate solution; the pH value of the mobile phase is preferably 1.0-5.0, and the best pH value is 2.0; the optimal detection wavelength is 260 nm; the best concentration of the test solution containing the cobimel is 0.2mg/mL, and the best injection volume is 10 mu L; the flow rate of the mobile phase is preferably 1.0 mL/min.

Has the advantages that: the invention utilizes convenient and fast high performance liquid chromatography to detect the Bomei, has good repeatability, accuracy and stability, particularly has stable retention time of chromatographic peak of the Bomei, and is beneficial to the quality control of medicines.

Drawings

FIG. 1 is a high performance liquid chromatogram of a blank solvent, acetonitrile;

FIG. 2 is a high performance liquid chromatogram of a system adaptation solution; peaks (1), (2), (3), (4), (5), (6), (7), (8), (9) are impurity 5, impurity 3, cobimel, impurity 2, starting material 1, intermediate II, intermediate I, impurity 4 and impurity 1, respectively.

FIG. 3 is a high performance liquid chromatogram of a test solution; peaks (1), (2) are impurity 5 and cobume, respectively.

Detailed Description

The following examples illustrate the invention but do not limit it in any way.

The instrument comprises the following steps: a set of simple high performance liquid chromatograph comprises a liquid phase pump, an ultraviolet detector and a sample injector.

Reagent: phosphoric acid (analytically pure), acetonitrile (chromatographically pure);

a chromatographic column: octadecyl bonded silica gel chromatographic column.

Detection wavelength: 260 nm;

chromatographic conditions are as follows: octadecyl bonded silica gel is used as a filling agent; 0.1% phosphoric acid solution was used as mobile phase A, acetonitrile was used as mobile phase B, the elution procedure is shown in Table 1, the detection wavelength was 260nm, the flow rate was 1.0mL/min, and the column temperature was 30 ℃.

TABLE 1 gradient elution procedure

Systematic adaptability test (high performance liquid chromatography, measured by 0512 of the four-part general regulation of the national pharmacopoeia 2015 edition) a proper amount of the cobume and the starting material 1, the intermediate I, the intermediate II, the impurity 1, the impurity 2, the impurity 3, the impurity 4 and the impurity 5 are taken, dissolved and diluted by acetonitrile to prepare a mixed solution containing about 0.2mg of the cobume, 0.2 mu g of the starting material 1, 0.2 mu g of the intermediate I, 0.2 mu g of the intermediate II, 0.2 mu g of the impurity 1, 0.2 mu g of the impurity 2, 0.2 mu g of the impurity 3, 0.2 mu g of the impurity 4 and 0.2 mu g of the impurity 5 in each 1mL, and the mixed solution is used as a systematic adaptability solution. Precisely measuring blank solution (namely acetonitrile) and system adaptability solution, respectively 10 μ L, injecting into a liquid chromatograph, recording chromatogram, as shown in figure 1 and figure 2, wherein the chromatogram shows that impurities and main peaks can be separated, and through calculation, the separation degree between each impurity and the main peak is greater than 1.5, according to the Bomei of the main peak, the tailing factor is less than 1.5, and the number of theoretical plates is greater than 5000.

Measuring Colume, precisely weighing Colume about 20mg, placing into a 100mL measuring flask, adding acetonitrile to dissolve and dilute to scale, shaking, and filtering to obtain test solution; precisely transferring 1.0mL of the test solution, placing the test solution in a 100mL measuring flask, adding acetonitrile to dilute the test solution to a scale, and shaking up to obtain a 1% self-control solution; injecting blank solution (acetonitrile), self-contrast solution and sample solution into liquid chromatograph, recording chromatogram, and calculating content of Colume and each impurity peak according to self-contrast method, as shown in figure 3.

Claims

1. A high performance liquid analysis method of Colubmet, characterized by comprising the steps of:

3) injecting the sample solution obtained in the step 1) into a high performance liquid chromatograph, wherein the sample injection volume is 1-100 mu L, and recording a chromatogram;

the volume content of acetonitrile in the mobile phase is 10-90%; the volume content of the phosphoric acid solution or the phosphate solution is 10 to 90 percent;

when the mobile phase is eluted, gradient elution is adopted, and the procedure of the gradient elution is as follows: changing the volume content of the phosphoric acid solution or the phosphate solution from 60% to 20% and the volume content of the acetonitrile from 40% to 80% for 0-20 min; 20-25min, keeping the volume content of the phosphoric acid solution or the phosphate solution at 20%, and keeping the volume content of the acetonitrile at 80%; changing the volume content of the phosphoric acid solution or the phosphate solution from 20% to 60% and the volume content of the acetonitrile from 80% to 40% within 25-27 min; 27-35min, the volume content of the phosphoric acid solution or the phosphate solution is kept at 60%, and the volume content of the acetonitrile is kept at 40%;

the pH value of the mobile phase is 1.0-5.0.

2. The method for HPLC analysis of Cobomet according to claim 1, wherein the volume concentration of phosphoric acid in said phosphoric acid solution is 0.01 to 10%.

3. The method for high performance liquid chromatography of cabomel according to claim 1, wherein the phosphate solution is one or more of potassium dihydrogen phosphate solution, sodium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, disodium hydrogen phosphate solution, potassium phosphate solution, and sodium phosphate solution.

4. The method for high performance liquid chromatography of cobimer according to claim 1, wherein the detection wavelength is 260 nm.

5. The method for HPLC analysis of Colbamet according to claim 1, wherein the test solution contains Colbamet at a concentration of 0.2mg/mL and a sample volume of 10. mu.L.

6. The method for high performance liquid chromatography of cobume according to claim 1, wherein the flow rate of the mobile phase is 1.0 mL/min.