CN103675185A - Method for determining all-trans isomers in entecavir tablets by high performance liquid chromatography - Google Patents

Abstract

The invention relates to a method for determining all-trans isomers in entecavir tablets by high performance liquid chromatography. According to the method, the content of the all-trans isomers of entecavir can be determined quantitatively by using a polysaccharide-coated chromatographic column and taking isopropanol-absolute ethyl alcohol-trifluoroacetic acid solution as a flow phase, wherein the volume ratio of the isopropanol, the absolute ethyl alcohol to the trifluoroacetic acid is 500:500:1. Therefore, the quality of medicaments can be controlled effectively.

Description

A kind of method with alltrans isomeride in high effective liquid chromatography for measuring Entecavir sheet

Technical field

The present invention relates to the detection technique field of Entecavir sheet, particularly a kind of method with alltrans isomeride in high effective liquid chromatography for measuring Entecavir sheet.

Background technology

Chronic hbv-infection is a difficult problem for global range medical science always, and exploitation anti-hepatic-B virus medicine is also a medical science focus always.Entecavir chemistry 2-amino-9-[(1S by name, 3R, 4S)-4-hydroxyl-3-methylol-2-methylene cyclopentyl]-1,9-dihydro-6H-purine-6-one monohydrate, is for guanosine analog, inhibited to hepatitis-B virus polymerase.Entecavir sheet is applicable to that virus replication is active, serum alanine aminotransferase (ALT) continues to raise or the treatment of chronic adult's hepatitis B patient that liver histological show events venereal disease becomes.

Entecavir has 3 chiral centers, may produce a plurality of optical isomer impurity, and alltrans isomeride can not detect under related substance inspection condition.

It is separated that the present invention adopts chiral chromatographic column to carry out Entecavir and alltrans isomeride, and detection method is optimized, thereby set up the detection method of alltrans isomeride in more effective detection Entecavir sheet.In the Entecavir sheet of setting up about this research optimization, alltrans method of separating isomers has no report.

Summary of the invention

Technical matters to be solved by this invention is to provide for the existing many technical matterss of existing Entecavir chip detection method a kind of method with alltrans isomeride in high effective liquid chromatography for measuring Entecavir sheet.

Technical matters to be solved by this invention can be achieved through the following technical solutions:

A method with alltrans isomeride in high effective liquid chromatography for measuring Entecavir sheet, comprises the steps:

(1) system suitability solution preparation process

Take Entecavir alltrans isomeride 3.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol 40mL, ultrasonic making dissolved, and lets cool, and absolute ethyl alcohol is diluted to scale, shakes up as Entecavir alltrans isomeride storing solution;

(2) system suitability solution preparation process

Take the about 6.0mg of Entecavir standard items, put in the brown measuring bottle of 50mL, add the about 40mL of absolute ethyl alcohol, ultrasonic making dissolved, let cool, precision measures Entecavir alltrans isomeride storing solution 1.0mL prepared by step (1) and adds, and adds absolute ethyl alcohol and is diluted to scale, mix, as system suitability solution;

(3) need testing solution preparation process

Entecavir sheet is ground into fine powder, and it is appropriate that precision takes fine powder, and fine powder is transferred in measuring bottle completely, adds absolute ethyl alcohol and make to dissolve and constant volume, filters, and gets subsequent filtrate and make every 1mL approximately containing the solution of Entecavir 0.15mg, shakes up, and filters, as need testing solution;

(4) reference substance solution preparation process

Need testing solution prepared by step (1) is centrifugal, and in the need testing solution supernatant from centrifugal, precision measures 1.0mL in 100mL volumetric flask, adds absolute ethyl alcohol and is diluted to scale, shakes up, and filters, and obtains mass percent concentration and be 1.0% reference substance solution;

(5) mobile phase preparation process

By absolute ethyl alcohol 500ml and 500ml isopropyl alcohol, add 1ml trifluoroacetic acid, mix, ultrasonic 30 minutes, obtain;

(6) chromatographic system establishment step

Chromatographic column: polysaccharide coating-type chromatographic column 250*4.6mm, column temperature: 30 ℃, detecting device: UV-detector, 250nm, flow velocity: 1.0ml/min, sampling volume: 20 μ l;

(7) elution step

Isocratic elution

(8) analytic process step

According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure;

Precision measures in system suitability solution 20 μ l injection liquid chromatographies, and going out front and back, peak order is isomeride and Entecavir, and the degree of separation of isomeride and main peak should be not less than 2.0;

Precision measures reference substance solution 20 μ l and injects the liquid chromatograph meet system suitability requirement, regulates detection sensitivity, and the peak height that makes major component chromatographic peak is full scale 20%.Accurate blank solution and the need testing solution injection liquid chromatography that measures 20 μ l, records chromatogram again;

(9) calculation procedure

According to the chromatogram of step (8) record, by major component Self-control method, calculate alltrans isomeride in Entecavir sheet, obtain; Specific formula for calculation is as follows:

$\%\mathrm{impurity\; A}=\frac{A_{\mathrm{spl}}}{A_{\mathrm{std}}}$

Note: A _splisomeride peak area in=sample solution; A _std=1% own control solution main peak peak area.

The present invention can quantitative measurement Entecavir alltrans content of isomer, thereby effectively control drug quality.

Accompanying drawing explanation

Fig. 1 is the alltrans isomer structure figure of Entecavir.

Embodiment

Mode by specific embodiment of the invention given below can further be well understood to the present invention, but they are not limitation of the invention.

A method with alltrans isomeride in high effective liquid chromatography for measuring Entecavir sheet, comprises the steps:

(1) system suitability solution preparation process

Take Entecavir alltrans isomeride 3.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol 40mL, ultrasonic making dissolved, and lets cool, and absolute ethyl alcohol is diluted to scale, shakes up as Entecavir alltrans isomeride storing solution;

(2) system suitability solution preparation process

Take the about 6.0mg of Entecavir standard items, put in the brown measuring bottle of 50mL, add the about 40mL of absolute ethyl alcohol, ultrasonic making dissolved, let cool, precision measures Entecavir alltrans isomeride storing solution 1.0mL prepared by step (1) and adds, and adds absolute ethyl alcohol and is diluted to scale, mix, as system suitability solution;

(3) need testing solution preparation process

Entecavir sheet is ground into fine powder, and it is appropriate that precision takes fine powder, and fine powder is transferred in measuring bottle completely, adds absolute ethyl alcohol and make to dissolve and constant volume, filters, and gets subsequent filtrate and make every 1mL approximately containing the solution of Entecavir 0.15mg, shakes up, and filters, as need testing solution;

(4) reference substance solution preparation process

Need testing solution prepared by step (1) is centrifugal, and in the need testing solution supernatant from centrifugal, precision measures 1.0mL in 100mL volumetric flask, adds absolute ethyl alcohol and is diluted to scale, shakes up, and filters, and obtains mass percent concentration and be 1.0% reference substance solution;

(5) mobile phase preparation process

By absolute ethyl alcohol 500ml and 500ml isopropyl alcohol, add 1ml trifluoroacetic acid, mix, ultrasonic 30 minutes, obtain;

(6) chromatographic system establishment step

Chromatographic column: polysaccharide coating-type chromatographic column 250*4.6mm, column temperature: 30 ℃, detecting device: UV-detector, 250nm, flow velocity: 1.0ml/min, sampling volume: 20 μ l;

(7) elution step

Isocratic elution

(8) analytic process step

According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure;

Precision measures in system suitability solution 20 μ l injection liquid chromatographies, and going out front and back, peak order is isomeride and Entecavir, and the degree of separation of isomeride and main peak should be not less than 2.0;

Precision measures reference substance solution 20 μ l and injects the liquid chromatograph meet system suitability requirement, regulates detection sensitivity, and the peak height that makes major component chromatographic peak is full scale 20%.Accurate blank solution and the need testing solution injection liquid chromatography that measures 20 μ l, records chromatogram again;

(9) calculation procedure

According to the chromatogram of step (8) record, by major component Self-control method, calculate alltrans isomeride in Entecavir sheet, obtain; Specific formula for calculation is as follows:

$\%\mathrm{impurity\; A}=\frac{A_{\mathrm{spl}}}{A_{\mathrm{std}}}$

Note: A _splisomeride peak area in=sample solution; A _std=1% own control solution main peak peak area.

In Entecavir sheet, alltrans isomeride testing result is as table 1

Table 1: alltrans isomeride testing result in Entecavir sheet

From above detection Entecavir sheet, alltrans isomeride testing result can be found out, according to method provided by the invention, in system flexibility solution, can prove that to make alltrans isomeride and Entecavir major component obtain effectively separated, there is precision high, reproducible, the advantage such as degree of separation is good.

Claims (1)

1. by a method for alltrans isomeride in high effective liquid chromatography for measuring Entecavir sheet, it is characterized in that, comprise the steps:

(1) system suitability solution preparation process

Take Entecavir alltrans isomeride 3.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol 40mL, ultrasonic making dissolved, and lets cool, and absolute ethyl alcohol is diluted to scale, shakes up as Entecavir alltrans isomeride storing solution;

(2) system suitability solution preparation process

Take the about 6.0mg of Entecavir standard items, put in the brown measuring bottle of 50mL, add the about 40mL of absolute ethyl alcohol, ultrasonic making dissolved, let cool, precision measures Entecavir alltrans isomeride storing solution 1.0mL prepared by step (1) and adds, and adds absolute ethyl alcohol and is diluted to scale, mix, as system suitability solution;

(3) need testing solution preparation process

Entecavir sheet is ground into fine powder, and it is appropriate that precision takes fine powder, and fine powder is transferred in measuring bottle completely, adds absolute ethyl alcohol and make to dissolve and constant volume, filters, and gets subsequent filtrate and make every 1mL approximately containing the solution of Entecavir 0.15mg, shakes up, and filters, as need testing solution;

(4) reference substance solution preparation process

Need testing solution prepared by step (1) is centrifugal, and in the need testing solution supernatant from centrifugal, precision measures 1.0mL in 100mL volumetric flask, adds absolute ethyl alcohol and is diluted to scale, shakes up, and filters, and obtains mass percent concentration and be 1.0% reference substance solution;

(5) mobile phase preparation process

By absolute ethyl alcohol 500ml and 500ml isopropyl alcohol, add 1ml trifluoroacetic acid, mix, ultrasonic 30 minutes, obtain;

(6) chromatographic system establishment step

Chromatographic column: polysaccharide coating-type chromatographic column 250*4.6mm, column temperature: 30 ℃, detecting device: UV-detector, 250nm, flow velocity: 1.0ml/min, sampling volume: 20 μ l;

(7) elution step

Isocratic elution

(8) analytic process step

According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure;

Precision measures in system suitability solution 20 μ l injection liquid chromatographies, and going out front and back, peak order is isomeride and Entecavir, and the degree of separation of isomeride and main peak should be not less than 2.0;

Precision measures reference substance solution 20 μ l and injects the liquid chromatograph meet system suitability requirement, regulates detection sensitivity, and the peak height that makes major component chromatographic peak is full scale 20%.Accurate blank solution and the need testing solution injection liquid chromatography that measures 20 μ l, records chromatogram again;

(9) calculation procedure

According to the chromatogram of step (8) record, by major component Self-control method, calculate alltrans isomeride in Entecavir sheet, obtain; Specific formula for calculation is as follows:

$\%\mathrm{impurity\; A}=\frac{A_{\mathrm{spl}}}{A_{\mathrm{std}}}$

Note: A _splisomeride peak area in=sample solution; A _std=1% own control solution main peak peak area.