CN110687238B - Detection method of flumatinib mesylate related substances - Google Patents

Abstract

The invention provides an HPLC detection method of a flumatinib mesylate related substance. The detection condition of the method is a high performance liquid chromatograph, which comprises the following steps: and (3) applying the substance to be detected to a chromatographic column, performing gradient elution by using a mobile phase, detecting an eluted product, and positioning and quantifying. The method has the advantages of strong specificity, high sensitivity and accurate quantitative determination result, can comprehensively detect the flumatinib mesylate and related substances thereof, and improves the safety of the medicine.

Description

Detection method of flumatinib mesylate related substances

Technical Field

The invention relates to the technical field of drug analysis, in particular to a method for detecting a flumatinib mesylate related substance.

Background

The chemical name of flumatinib mesylate is 4- [ (4-methyl-1-piperazinyl) methyl ] -N- [ 6-methyl-5- [ [4- (3-pyridinyl) -2-pyrimidinyl ] amino ] pyridin-3-yl ] -3- (trifluoromethyl) -benzamide methanesulfonate.

In recent years, gleevec, also known as imatinib, has become the first-line drug for treating chronic myelogenous leukemia, but some patients develop drug resistance after use. In most cases, drug resistance occurs because BCR-ABL undergoes a mutation, resulting in a structural change in the enzyme, such that gleevec cannot bind to it. The flumatinib mesylate is a new generation of leukemia treatment medicine developed by Jiangsu Haoshen pharmaceutical industry group Limited company on the basis of imatinib, is mainly used for treating chronic granulocytic leukemia, and can better solve the problem of drug resistance.

Compared with gleevec, the flumatinib mesylate structurally introduces a proper group to improve the binding force with kinase, and is expected to improve the binding force with a kinase mutant. Meanwhile, in the aspect of treating chronic myelogenous leukemia, the drug effect of the flumatinib mesylate is obviously higher than that of gleevec, so that the flumatinib mesylate has wider market potential.

At present, the patent literature about the preparation of the flumatinib mesylate is less, and particularly, the patent literature about the research of a detection method of a substance related to the flumatinib mesylate is less.

The impurities of the flumatinib mesylate mainly come from potential impurities existing in the production and storage processes, including unreacted intermediates and raw materials, impurities generated in the reaction and impurities generated in the drug degradation. The impurity structure is as follows:

in order to ensure the safe use of the medicine, the quality control of the medicine is generally required, and the related substances are items which must be detected. At present, the quality standard of flumatinib mesylate is not collected in pharmacopoeias of various countries. Therefore, it is necessary to develop a method for detecting a substance.

Disclosure of Invention

The invention aims to provide a method for detecting a flumatinib mesylate related substance.

Specifically, the present invention provides:

a detection method of flumatinib mesylate related substances is characterized by comprising the following steps: the detection condition is a high performance liquid chromatograph, comprising: and (3) applying the substance to be detected to a chromatographic column, performing gradient elution by using a mobile phase, detecting an eluted product, and positioning and quantifying.

Further, the column is a C18 reverse phase column, including an agent SB C18, Phenomenex Luna C18 or Waters XBridge C18 column.

Furthermore, the filler of the chromatographic column is octadecyl bonded silica gel.

Further, the mobile phase consists of a mobile phase A and a mobile phase B; wherein the mobile phase A is a buffer solution, and the mobile phase B is an organic solvent.

Further, the mobile phase A also comprises a tail scavenging agent, preferably triethylamine; the mobile phase B is selected from methanol or acetonitrile, preferably acetonitrile.

Further, the triethylamine is added in an amount of 1% (by volume).

Further, the buffer solution has a pH of 2.0 to 4.0, preferably 3.0.

Further, the buffer solution is a phosphate buffer solution, and the phosphate is selected from one or more of sodium phosphate, potassium phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, dipotassium hydrogen phosphate and disodium hydrogen phosphate, and potassium dihydrogen phosphate is preferred.

Further, the concentration of the buffer solution is 0.02 to 0.08mol/L, preferably 0.05 mol/L. Further, the flow rate of the gradient elution is 0.8-1.2 mL/min, preferably 1.0mL/min

Further, the column temperature of the chromatographic column is 20 to 30 ℃, preferably 25 ℃.

Furthermore, the detection method adopts an external standard method to carry out positioning and quantification on related substances.

Furthermore, the detection wavelength of the detection method is 240-263 nm, and is preferably 242 nm.

Further, the procedure of the gradient elution is:

time 0min, mobile phase a: 90%, mobile phase B: 10 percent;

time 50min, mobile phase a: 80%, mobile phase B: 20 percent;

time 60min, mobile phase a: 70%, mobile phase B: 30 percent;

time 80min, mobile phase a: 70%, mobile phase B: 30 percent;

time 81min, mobile phase a: 90%, mobile phase B: 10 percent;

time 90min, mobile phase a: 90%, mobile phase B: 10 percent.

The method establishes a liquid phase analysis method aiming at impurities introduced and generated by degradation in a preparation process of flumatinib mesylate, adopts a conventional durable C18 chromatographic column, detects at low wavelength, performs elution by using an optimized gradient elution program, analyzes a large number of impurities, and can completely separate known impurities and unknown impurities degraded by destruction tests of acid, alkali, heat, oxygen, high temperature, illumination and the like.

Drawings

FIG. 1: high performance liquid chromatogram of the related substances of flumatinib mesylate.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

It will be understood by those skilled in the art, based upon the disclosure herein, that various modifications and improvements may be made to the invention without departing from the spirit and scope of the invention. They are intended to fall within the scope of protection of the patent as defined by the claims of the present application. Furthermore, it should be understood that the examples provided herein are for the purpose of illustrating the invention and should not be construed as limiting the invention.

The impurities A and B are known impurities, and the preparation method thereof is shown in CN201610595756.4 and CN201710621601.8.

Impurity C control preparation: dissolving flumatinib mesylate (10.0g) in hydrogen peroxide (50mL, content 10%), stirring at room temperature for 24h, adding saturated aqueous sodium bicarbonate solution (100mL), extracting with ethyl acetate 100mL × 5, synthesizing an organic phase, drying with anhydrous sodium sulfate, filtering, concentrating to obtain an oily substance, performing column chromatography, and obtaining a mobile phase: dichloromethane-methanol (20: 1) gave 0.5g of a solid.

Impurity D control preparation: flumatinib mesylate (10.0g) is dissolved in hydrogen peroxide (50mL, content 10%), and stirred for 72h at room temperature, so that the reaction solution is prepared, and 0.9g of the target compound is obtained.

Experiment 1 mobile phase Condition screening

Chromatographic conditions are as follows: a chromatographic column: phenomenex Luna (2)

4.6X 200mm, 5 μm; the flow rate is 1 mL/min; column temperature: 25 ℃; the detection wavelength is 242 nm; other conditions are shown in table 1 below.

Sample preparation: taking 50mg of flumatinib mesylate as a raw material, precisely weighing, placing in a 50mL volumetric flask, dissolving and diluting to a scale with a mobile phase A-mobile phase B (80: 20) solution, precisely weighing 20 mu L, and injecting into a liquid chromatograph.

TABLE 1 comparative testing of different flow conditions

According to experimental results, compared with isocratic elution, gradient elution can separate each impurity peak, and the screening method is high in sensitivity and good in separation degree. (see FIG. 1)

Experiment 2 investigation of influencing factors

TABLE 2 comparative testing of different influencing factor conditions

Mobile phase: mobile phase a was 0.05M potassium dihydrogen phosphate buffer plus 1% TEA (pH 3.0 adjusted with phosphoric acid), mobile phase B was acetonitrile; the flow rate is 1 mL/min; column temperature: 25 ℃; the detection wavelength was 242 nm.

And (3) an elution mode: gradient elution, elution procedure: mobile phase A: 0min (90%), 50min (80%), 60min (70%), 80min (70%), 81min (90%), 90mim (90%)

Sample preparation: taking 50mg of flumatinib mesylate raw material medicine, precisely weighing, placing in a 50mL volumetric flask, dissolving and diluting to a scale with a mobile phase A-mobile phase B (80: 20) solution, and carrying out sample injection analysis. The results are given in Table 3 below

TABLE 3 time to peak of each peak under different chromatographic column conditions

Each impurity peak can be completely separated from the main peak, the separation degree of the flumatinib mesylate and the imatinib mesylate is far greater than 1.5, and the two components are completely separated.

Experiment 3 impurity recovery rate matrix sample preparation: weighing 1g of flumatinib mesylate, placing the flumatinib mesylate in a 100mL measuring flask, dissolving the flumatinib mesylate with a proper amount of acetonitrile, and dissolving and diluting the solution of the mobile phase A-the mobile phase B (80: 20) to a scale mark to obtain a recovery matrix sample.

Preparing an impurity diluent: weighing about 10mg of each of the A, B, C, D reference substances, placing the A, B, C, D reference substances into a 100mL measuring flask, dissolving with an appropriate amount of acetonitrile, dissolving with a mobile phase A-mobile phase B (80: 20) solution, diluting to scale, and preparing three parts in parallel for later use.

Preparing a test article: the above-mentioned impurity dilutions were precisely measured in an amount of 0.4mL, 1mL and 2mL, respectively, based on 100% of the measured concentration of 0.1% of each impurity limit, and placed in a 100mL measuring flask containing 1g of the matrix sample, and dissolved with a mobile phase diluent to a constant volume, and the solutions were prepared in triplicate to obtain sample solutions of 40%, 100% and 200% of each impurity limit, respectively.

Preparing a reference substance: 1mL of the impurity diluted solution was precisely measured and placed in a 100mL measuring flask as a control solution.

Chromatographic conditions are as follows: a chromatographic column: phenomenex Luna (2)

4.6X 200mm, 5 μm; the flow rate is 1 mL/min; column temperature: 25 ℃; mobile phase: mobile phase a was 0.05M potassium dihydrogen phosphate buffer plus 1% TEA (pH 3.0 adjusted with phosphoric acid), mobile phase B was acetonitrile; the flow rate is 1 mL/min; column temperature: 25 ℃; the detection wavelength was 242 nm. And (3) an elution mode: gradient elution, elution procedure: mobile phase A: 0min (90%), 50min (80%), 60min (70%), 80min (70%), 81min (90%), 90mim (90%)

Precisely measuring 20 mu L of each sample at the wavelength of 242nm, injecting the sample into a chromatograph, recording a chromatogram, measuring a peak area, and calculating the recovery rate by adopting an external standard method, wherein the recovery rate result is shown in the table.

TABLE 4 determination of the recovery of impurity A

TABLE 5 determination of the recovery of impurity B

TABLE 6 measurement results of recovery of impurity C

TABLE 7 determination of the recovery of impurity D

As can be seen from the above series of tables, the detection method detects the impurity A, B, C, D with good accuracy.

Experiment 4 verification of detection limit and quantification limit

Under the same conditions as in experiment 3, the detection limit of flumatinib mesylate and each impurity was calculated as S/N ═ 3, and the quantification limit of flumatinib mesylate and each impurity was calculated as S/N ═ 10. The results of the experiment are shown in the following table:

Claims (1)

1. a detection method of flumatinib mesylate related substances is characterized in that the detection method can simultaneously detect 4 impurities, and the structures of the impurities are as follows:

the detection method comprises the steps of loading a substance to be detected on a chromatographic column, performing gradient elution by using a mobile phase, and performing HPLC (high performance liquid chromatography) determination on an eluted product;

the chromatographic column is an Agient SB C18, Phenomenex Luna C18 or Waters Xbridge C18 chromatographic column; the mobile phase consists of a mobile phase A and a mobile phase B; wherein the mobile phase A is potassium dihydrogen phosphate buffer solution, and the mobile phase B is acetonitrile; the mobile phase A also comprises a tail scavenging agent triethylamine, and the addition amount of the triethylamine is 1%; the pH of the mobile phase a is 3.0; the concentration of the potassium dihydrogen phosphate buffer solution is 0.05 mol/L;

the flow rate of the gradient elution is 1.0 mL/min; the column temperature of the chromatographic column is 25 ℃; the detection wavelength is 242 nm;

the procedure for the gradient elution was:

time 0min, mobile phase a: 90%, mobile phase B: 10 percent;

time 50min, mobile phase a: 80%, mobile phase B: 20 percent;

time 60min, mobile phase a: 70%, mobile phase B: 30 percent;

time 80min, mobile phase a: 70%, mobile phase B: 30 percent;

time 81min, mobile phase a: 90%, mobile phase B: 10 percent;

time 90min, mobile phase a: 90%, mobile phase B: 10 percent.

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