CN103675185B - A kind of method of all trans isomer in high effective liquid chromatography for measuring Entecavir tablet - Google Patents
A kind of method of all trans isomer in high effective liquid chromatography for measuring Entecavir tablet Download PDFInfo
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- CN103675185B CN103675185B CN201310666403.5A CN201310666403A CN103675185B CN 103675185 B CN103675185 B CN 103675185B CN 201310666403 A CN201310666403 A CN 201310666403A CN 103675185 B CN103675185 B CN 103675185B
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Abstract
The present invention relates to a kind of method of liquid chromatography for separating and determining Entecavir and all trans isomer thereof, the method is with polysaccharide coating-type chromatographic column, with isopropyl alcohol-absolute ethyl alcohol-trifluoroacetic acid solution for mobile phase, isopropyl alcohol-absolute ethyl alcohol-trifluoroacetic acid volume ratio is 500: 500: 1, can all trans isomer content of quantitative measurement Entecavir, thus effectively control drug quality.
Description
Technical field
The present invention relates to the detection technique field of Entecavir tablet, particularly a kind of method of all trans isomer in high effective liquid chromatography for measuring Entecavir tablet.
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-methylenecyclopentyl]-1,9-dihydro-6H-purine-6-one monohydrate, be guanosine analog, inhibited to hepatitis-B virus polymerase.Entecavir tablet is applicable to the treatment of chronic adult's hepatitis B patient that virus replication is active, serum alanine aminotransferase (ALT) continues rising or liver histological show events venereal disease becomes.
Entecavir has 3 chiral centers, and may produce multiple optical isomer impurity, all trans isomer can not detect under Related substances separation condition.
The present invention adopts chiral chromatographic column to be separated with all trans isomer Entecavir, and is optimized detection method, thus establishes the detection method of all trans isomer in more effective detection Entecavir tablet.Report is had no about all trans isomer separation method in the Entecavir tablet that this research optimization is set up.
Summary of the invention
Technical matters to be solved by this invention is the method providing all trans isomer in a kind of high effective liquid chromatography for measuring Entecavir tablet for the many technical matterss existing for existing Entecavir chip detection method.
Technical matters to be solved by this invention can be achieved through the following technical solutions:
In high effective liquid chromatography for measuring Entecavir tablet, a method for all trans isomer, comprises the steps:
(1) system suitability solution preparation step
Take Entecavir all trans isomer 3.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol 40mL, ultrasonicly make dissolving, let cool, absolute ethyl alcohol is diluted to scale, shakes up as Entecavir all trans isomer storing solution;
(2) system suitability solution preparation step
Take Entecavir standard items and be about 6.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol and be about 40mL, ultrasonicly make dissolving, let cool, precision measures Entecavir all trans isomer storing solution 1.0mL prepared by step (1) and adds, and adds absolute ethyl alcohol and is diluted to scale, mixing, as system suitability solution;
(3) need testing solution preparation process
Entecavir tablet is ground into fine powder, and it is appropriate that precision takes fine powder, is transferred to completely in measuring bottle by fine powder, adds absolute ethyl alcohol and make dissolving and constant volume, filters, and gets subsequent filtrate and make every 1mL about containing the solution of Entecavir 0.15mg, shake up, filter, as need testing solution;
(4) reference substance solution preparation process
Need testing solution step (1) prepared is centrifugal, from the need testing solution supernatant after centrifugal, precision measures in 1.0mL to 100mL volumetric flask, adds absolute ethyl alcohol and is diluted to scale, shake up, filter, obtain the reference substance solution that mass percent concentration is 1.0%;
(5) mobile phase preparation process
By absolute ethyl alcohol 500ml and 500ml isopropyl alcohol, add 1ml trifluoroacetic acid, mixing, ultrasonic 30 minutes, to obtain final product;
(6) chromatographic system establishment step
Chromatographic column: polysaccharide coating-type chromatographic column 250*4.6mm, column temperature: 30 DEG C, detecting device: UV-detector, 250nm, flow velocity: 1.0ml/min, sampling volume: 20 μ l;
(7) elution step
Isocratic elution
(8) analytic process step
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 two annex V D);
Precision measures in system suitability solution 20 μ l injection liquid chromatography, and going out peak tandem 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 meeting system suitability requirement, regulates detection sensitivity, makes the peak height of major component chromatographic peak be 20% of full scale.Accurate blank solution and the need testing solution injection liquid chromatography measuring 20 μ l again, record chromatogram;
(9) calculation procedure
According to the chromatogram that step (8) records, calculate all trans isomer in Entecavir tablet by major component Self-control method, to obtain final product; Specific formula for calculation is as follows:
Note: A
splisomeride peak area in=sample solution; A
std=1% own control solution main peak peak area.
The present invention can all trans isomer content of quantitative measurement Entecavir, thus effectively control drug quality.
Accompanying drawing explanation
Fig. 1 is all trans isomer structural drawing of Entecavir.
Embodiment
Can be well understood to the present invention further by the mode of specific embodiment of the invention given below, but they not limitation of the invention.
In high effective liquid chromatography for measuring Entecavir tablet, a method for all trans isomer, comprises the steps:
(1) system suitability solution preparation step
Take Entecavir all trans isomer 3.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol 40mL, ultrasonicly make dissolving, let cool, absolute ethyl alcohol is diluted to scale, shakes up as Entecavir all trans isomer storing solution;
(2) system suitability solution preparation step
Take Entecavir standard items and be about 6.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol and be about 40mL, ultrasonicly make dissolving, let cool, precision measures Entecavir all trans isomer storing solution 1.0mL prepared by step (1) and adds, and adds absolute ethyl alcohol and is diluted to scale, mixing, as system suitability solution;
(3) need testing solution preparation process
Entecavir tablet is ground into fine powder, and it is appropriate that precision takes fine powder, is transferred to completely in measuring bottle by fine powder, adds absolute ethyl alcohol and make dissolving and constant volume, filters, and gets subsequent filtrate and make every 1mL about containing the solution of Entecavir 0.15mg, shake up, filter, as need testing solution;
(4) reference substance solution preparation process
Need testing solution step (1) prepared is centrifugal, from the need testing solution supernatant after centrifugal, precision measures in 1.0mL to 100mL volumetric flask, adds absolute ethyl alcohol and is diluted to scale, shake up, filter, obtain the reference substance solution that mass percent concentration is 1.0%;
(5) mobile phase preparation process
By absolute ethyl alcohol 500ml and 500ml isopropyl alcohol, add 1ml trifluoroacetic acid, mixing, ultrasonic 30 minutes, to obtain final product;
(6) chromatographic system establishment step
Chromatographic column: polysaccharide coating-type chromatographic column 250*4.6mm, column temperature: 30 DEG C, detecting device: UV-detector, 250nm, flow velocity: 1.0ml/min, sampling volume: 20 μ l;
(7) elution step
Isocratic elution
(8) analytic process step
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 two annex V D);
Precision measures in system suitability solution 20 μ l injection liquid chromatography, and going out peak tandem 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 meeting system suitability requirement, regulates detection sensitivity, makes the peak height of major component chromatographic peak be 20% of full scale.Accurate blank solution and the need testing solution injection liquid chromatography measuring 20 μ l again, record chromatogram;
(9) calculation procedure
According to the chromatogram that step (8) records, calculate all trans isomer in Entecavir tablet by major component Self-control method, to obtain final product; Specific formula for calculation is as follows:
Note: A
splisomeride peak area in=sample solution; A
std=1% own control solution main peak peak area.
In Entecavir tablet, all trans isomer testing result is as table 1
Table 1: all trans isomer testing result in Entecavir tablet
All trans isomer testing result in Entecavir tablet is detected as can be seen from above, according to method provided by the invention, can prove in system flexibility solution to make all trans isomer be effectively separated with Entecavir major component, there is precision high, reproducible, the advantages such as degree of separation is good.
Claims (1)
1., by a method for all trans isomer in high effective liquid chromatography for measuring Entecavir tablet, it is characterized in that, comprise the steps:
(1) system suitability solution preparation step
Take Entecavir all trans isomer 3.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol 40mL, ultrasonicly make dissolving, let cool, absolute ethyl alcohol is diluted to scale, shakes up as Entecavir all trans isomer storing solution;
Take Entecavir standard items and be about 6.0mg, put in the brown measuring bottle of 50mL, add absolute ethyl alcohol and be about 40mL, ultrasonicly make dissolving, let cool, precision measures Entecavir all trans isomer storing solution 1.0mL prepared by step (1) and adds, and adds absolute ethyl alcohol and is diluted to scale, mixing, as system suitability solution;
(2) need testing solution preparation process
Entecavir tablet is ground into fine powder, and it is appropriate that precision takes fine powder, is transferred to completely in measuring bottle by fine powder, adds absolute ethyl alcohol and make dissolving and constant volume, filters, and gets subsequent filtrate and make every 1mL about containing the solution of Entecavir 0.15mg, shake up, filter, as need testing solution;
(3) 1.0% own control solution preparation step
Need testing solution step (2) prepared is centrifugal, and from the need testing solution supernatant after centrifugal, precision measures in 1.0mL to 100mL volumetric flask, adds absolute ethyl alcohol and is diluted to scale, shake up, filter and get final product;
(4) mobile phase preparation process
By absolute ethyl alcohol 500ml and 500ml isopropyl alcohol, add 1ml trifluoroacetic acid, mixing, ultrasonic 30 minutes, to obtain final product;
(5) chromatographic system establishment step
Chromatographic column: polysaccharide coating-type chromatographic column 250 × 4.6mm, column temperature: 30 DEG C, detecting device: UV-detector, 250nm, flow velocity: 1.0ml/min, sampling volume: 20 μ l;
(6) elution step
Isocratic elution
(7) analytic process step
According to the high effective liquid chromatography for measuring that Chinese Pharmacopoeia version in 2010 two annex V D announce;
Precision measures in system suitability solution 20 μ l injection liquid chromatography, and going out peak tandem is isomeride and Entecavir, and the degree of separation of isomeride and main peak should be not less than 2.0;
Precision measures 1.0% own control solution 20 μ l and injects the liquid chromatograph meeting system suitability requirement, regulates detection sensitivity, makes the peak height of major component chromatographic peak be 20% of full scale; Accurate blank solution and the need testing solution injection liquid chromatography measuring 20 μ l again, record chromatogram;
(8) calculation procedure
According to the chromatogram that step (7) records, calculate all trans isomer in Entecavir tablet by major component Self-control method, to obtain final product.
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| KR101150254B1 (en) * | 2008-12-26 | 2012-06-12 | 한미사이언스 주식회사 | Process for preparing entecavir and intermediates used therein |
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Address after: 200120 Shanghai city Baoshan District Luo Road No. 50 Patentee after: Shanghai Jingfeng Pharmaceutical Co., Ltd. Address before: 200120 Shanghai city Baoshan District Luo Road No. 50 Patentee before: Shanghai Jingfeng Pharmaceutical Co., Ltd. |