CN114324665B

CN114324665B - Detection method of mutation-causing impurities of adefovir dipivoxil tablets

Info

Publication number: CN114324665B
Application number: CN202111670042.2A
Authority: CN
Inventors: 孔凡刚; 程红艳; 杜爽; 李续
Original assignee: Cisen Pharmaceutical Co ltd
Current assignee: Cisen Pharmaceutical Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-02-24
Anticipated expiration: 2041-12-30
Also published as: CN114324665A

Abstract

The invention discloses a method for detecting mutation-causing impurities of adefovir dipivoxil tablets. The detection method comprises the following steps: precisely measuring the reference solution and the test solution, respectively injecting into a liquid chromatograph, recording chromatogram, and calculating the content of benzyl chloride by peak area according to an external standard method. The invention separates out the mutagenic impurities of the adefovir dipivoxil tablets, thereby realizing the separation and the determination of other impurities and the mutagenic impurities in the adefovir dipivoxil tablets and realizing the quality control of the preparation. The invention adopts a gradient elution mode with simple operation, and has short operation time, high sensitivity, and good separation degree and reproducibility.

Description

Detection method of mutation-causing impurities of adefovir dipivoxil tablets

Technical Field

The invention belongs to the technical field of medicine quality detection, and particularly relates to a method for detecting mutation-causing impurities of adefovir dipivoxil tablets.

Background

The adefovir dipivoxil tablet is white or white-like tablet, and is suitable for treating adult chronic hepatitis B patients with hepatitis B virus activity replication evidence, continuous increase of serum amino acid transferase (ALT or AST) or liver function compensation of liver histology activity lesion. The hydroxymethyl impurity structure contains mutation-causing impurities with N-hydroxymethyl warning structures, and is mainly generated by degradation of adefovir monoester. The adefovir monoester is the main degradation impurity of the adefovir dipivoxil, and the adefovir monoester and the degradation product formaldehyde thereof react to generate hydroxymethyl impurities. The generation process is as follows:

disclosure of Invention

The invention aims to provide a method for detecting mutation-causing impurities of adefovir dipivoxil tablets.

The detection method of the mutation-causing impurities of the adefovir dipivoxil tablet comprises the following steps: precisely measuring a reference solution and a test solution, respectively injecting the reference solution and the test solution into a liquid chromatograph, recording a chromatogram, and calculating the content of benzyl chloride by peak area according to an external standard method; the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler; taking 0.02-0.05mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B; the detection wavelength is 210-280nm; the flow rate is 0.8-1.1mL per minute; the column temperature is 25-40 ℃; injection volume 5-20 μ l, elution gradient:

the preparation method of the test solution comprises the following steps: grinding a medicine to be detected into fine powder, accurately weighing, placing the fine powder in a 50ml measuring flask, adding 40ml of a solvent, carrying out ice bath and ultrasonic treatment for 15 minutes to dissolve adefovir dipivoxil, placing the solution to room temperature, diluting the solution to a scale with the solvent, shaking up, filtering, and taking a subsequent filtrate; the solvent is a mixed solution of potassium dihydrogen phosphate solution and acetonitrile with a volume ratio of 82, wherein the concentration of the potassium dihydrogen phosphate solution is 0.025mol/L.

The preparation method of the reference substance solution comprises the following steps: precisely weighing a hydroxymethyl impurity reference substance, adding a solvent for dissolving, and quantitatively diluting to prepare a solution containing 2 microgrammes of hydroxymethyl impurities in 1ml; the solvent is a mixed solution of potassium dihydrogen phosphate solution and acetonitrile with a volume ratio of 82, wherein the concentration of the potassium dihydrogen phosphate solution is 0.025mol/L.

The invention separates out the mutagenic impurities of the adefovir dipivoxil tablets, thereby realizing the separation and the determination of other impurities and the mutagenic impurities in the adefovir dipivoxil tablets and realizing the quality control of the preparation. The invention adopts a gradient elution mode with simple operation, and has short operation time, high sensitivity, and good separation degree and reproducibility.

Drawings

FIG. 1 is a detection profile obtained in accordance with an embodiment.

Detailed Description

Preparing each solution used in the detection:

solvent: 0.025mol/L potassium dihydrogen phosphate solution-acetonitrile (82;

test solution: taking a proper amount of fine powder (about equivalent to 50mg of adefovir dipivoxil) of the product, accurately weighing, placing in a 50ml measuring flask, adding about 40ml of solvent, carrying out ultrasonic treatment in ice bath for 15 minutes to dissolve the adefovir dipivoxil, placing to room temperature, diluting to scale with the solvent, shaking up, filtering, and taking a subsequent filtrate.

Control solution: taking a proper amount of hydroxymethyl impurity reference substance, precisely weighing, adding a solvent to dissolve, and quantitatively diluting to prepare a solution containing 2 mu g of hydroxymethyl impurity in each 1 ml.

System applicability solution: respectively taking appropriate amount of adefovir monoester and hydroxymethyl impurity reference substance, dissolving with solvent, diluting to obtain solution containing adefovir monoester 25 μ g and hydroxymethyl impurity 2 μ g in each 1ml, and ultrasonic dissolving in ice bath if necessary.

Sensitivity solution: taking a proper amount of adefovir dipivoxil reference substance, adding a solvent to dissolve and dilute the adefovir dipivoxil reference substance to prepare a solution containing about 0.1 mu g of adefovir dipivoxil in each 1 ml.

Blank auxiliary materials: precisely weighing 700mg of blank auxiliary materials, placing the blank auxiliary materials into a 50ml measuring flask, adding about 40ml of diluent, carrying out ice bath ultrasonic treatment for 15 minutes, placing the mixture to room temperature, diluting the mixture to a scale with the diluent, shaking up the mixture, filtering the mixture, and taking the subsequent filtrate to obtain the product.

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; gradient elution is carried out by taking 0.02-0.05mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B; the detection wavelength is 210-280nm; the flow rate is 0.8-1.1ml per minute; the column temperature is 25-40 ℃; the sample injection volume is 5-20 mul; gradient elution conditions:

time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	95	5
2	95	5
			4	82	18
12	60	40
			21	60	40
28	30	70
			38	30	70
42	95	5
			50	95	5

System applicability requirements: in a system applicability solution chromatogram, the separation degree between hydroxymethyl impurities and an adefovir monoester peak is more than 1.5; in a sensitive solution chromatogram, the signal-to-noise ratio of the peak height of the adefovir dipivoxil is greater than 10.

The determination method comprises the following steps: precisely measuring the test solution and the reference solution, respectively injecting into a liquid chromatograph, and recording the chromatogram.

Results of the specificity experiment

The blank solvent and the blank auxiliary materials have no interference on the detection of hydroxymethyl impurities, the separation degree is greater than 1.5, and the specificity experiment is passed.

Preparing a quantitative limit solution and a detection limit solution:

quantitative limiting solution:

detection limiting solution:

experimental results of limit of detection

Detection limit concentration (ng/ml)	Detection limit (ng)	S/N
			5.269	0.005269	6.8

Quantitative limit test results

The detection limit and the quantitative limit experiment result can meet the detection requirement.

Linear and range solution formulation:

hydroxymethyl impurity stock solution:

hydroxymethyl impurity limit of quantitation solution:

linear 1

Linearity 2

Linear 3

Linearity 4

Linear 5

Results of linearity and range experiments

Preparing an accurate solution:

hydroxymethyl impurity stock solution:

control solution:

blank test article solution: weighing 750mg of the fine powder, placing the fine powder into a 50ml measuring flask, adding about 40ml of diluent, carrying out ice bath ultrasonic treatment for 15 minutes, placing the flask to room temperature, diluting the flask to a scale with the diluent, shaking up, filtering, and taking subsequent filtrate to obtain 3 parts in parallel.

Accuracy solution: accurately weighing 750mg and 9 parts of fine powder of the product, respectively placing the fine powder into 50ml measuring bottles, respectively accurately adding 1.6ml, 2.0ml and 2.4ml of hydroxymethyl impurity stock solution into 3 parts of fine powder, respectively adding about 40ml of diluent, carrying out ice bath ultrasonic treatment for 15 minutes, standing to room temperature, diluting to a scale mark by using the diluent, shaking uniformly, filtering, and taking subsequent filtrate to obtain the product.

Accuracy experimental results

The average recovery of hydroxymethyl impurities (n = 9) resulted in 103.2% and RSD 2.4%, with accuracy verification results meeting the requirements.

Preparing a repetitive solution:

hydroxymethyl impurity stock solution:

control solution:

Repetitive solution: accurately weighing 750mg and 6 parts of fine powder of the product, respectively placing into 50ml measuring bottles, accurately adding 2.0ml of hydroxymethyl impurity stock solution respectively, adding about 40ml of diluent, carrying out ice bath and ultrasonic treatment for 15 minutes, standing to room temperature, diluting to scale with diluent, shaking uniformly, filtering, and taking subsequent filtrate to obtain the product.

Results of repeatability experiments

The repetitive results meet the detection requirements.

Claims

1. The detection method of the mutation-causing impurities of the adefovir dipivoxil tablet is characterized in that the mutation-causing impurities are hydroxymethyl impurities of the adefovir monoester, and the structural formula of the mutation-causing impurities is as follows:

the detection method is specifically operative to: precisely measuring a reference solution and a test solution, respectively injecting the reference solution and the test solution into a liquid chromatograph, recording a chromatogram, and calculating the content of hydroxymethyl impurities of the adefovir monoester according to an external standard method by peak area; the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler; taking 0.02-0.05mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B; the detection wavelength is 210-280nm; the flow rate is 0.8-1.1mL per minute; the column temperature is 25-40 ℃; injection volume is 5-20 μ L, elution gradient is:

。

2. The method for detecting mutagenic impurities in adefovir dipivoxil tablets according to claim 1, wherein the preparation method of the test solution comprises: grinding a drug to be tested into fine powder, accurately weighing, placing the fine powder in a 50ml measuring flask, adding 40ml of a solvent, carrying out ultrasonic treatment in an ice bath for 15 minutes to dissolve adefovir dipivoxil, placing the solution to room temperature, diluting the solution to a scale with the solvent, shaking up, filtering, and taking a subsequent filtrate; the solvent is a mixed solution of potassium dihydrogen phosphate solution and acetonitrile with a volume ratio of 82, wherein the concentration of the potassium dihydrogen phosphate solution is 0.025mol/L.

3. The method for detecting the mutagenic impurities in the adefovir dipivoxil tablets as claimed in claim 1, wherein the preparation method of the reference solution comprises: precisely weighing a hydroxymethyl impurity reference substance, adding a solvent to dissolve and quantitatively diluting to prepare a solution containing 2 microgram of hydroxymethyl impurities in each 1ml; the solvent is a mixed solution of potassium dihydrogen phosphate solution and acetonitrile with a volume ratio of 82, wherein the concentration of the potassium dihydrogen phosphate solution is 0.025mol/L.