CN101701942A - Method for separating and measuring entecavir and optical isomer thereof by liquid chromatography - Google Patents
Method for separating and measuring entecavir and optical isomer thereof by liquid chromatography Download PDFInfo
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- CN101701942A CN101701942A CN200910104763A CN200910104763A CN101701942A CN 101701942 A CN101701942 A CN 101701942A CN 200910104763 A CN200910104763 A CN 200910104763A CN 200910104763 A CN200910104763 A CN 200910104763A CN 101701942 A CN101701942 A CN 101701942A
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Abstract
The invention relates to a method for separating and measuring entecavir and optical isomer thereof by liquid chromatography. The method takes 3 (3, 5-dimethyl-phenyl)-carbamate amylase as chiral chromatographic column of filler and hexane - lower alcohol solution as flowing phase, and the volume ratio of the hexane - lower alcohol solution is 25:75-75:25. Furthermore, the method can be used for measuring the content of the optical isomer of the entecavir in a quantitative way, thus effectively controlling the product quality.
Description
Technical field
The invention belongs to the analytical chemistry field, be specifically related to method with liquid chromatography for separating and determining Entecavir and optical isomer thereof.
Background technology
Entecavir is a kind of anti-hepatitis virus medicine, is mainly used in to suppress duplicating of hepatitis B, and its molecular formula is C
12H
15N
5O
3., its structural formula is:
Contain 3 asymmetric carbon atoms in the Entecavir molecule, generate different chiral isomers, in the medicine building-up process, need optical isomer is carried out quality control by asymmetric syntheses.Containing the separation of enantiomter of a plurality of asymmetric carbon atoms known is the difficult point of quality control in the synthetic and preparation process of chiral drug, therefore, realize having important practical significance the synthesizing of Entecavir that be separated in of Entecavir and chiral isomer thereof with the research of preparation, the quality control aspect of production run.
Younger sister Jiang Yin in 2008: " formulation and technology of entecavir dispersible tablet and quality standard research " (Shenyang Pharmaceutical University's academic dissertation), disclosing with Daicel ChiralpakAD-H chiral chromatographic column is separating column, with absolute ethyl alcohol (containing 0.1% trifluoroacetic acid)-normal hexane (40: 60) is moving phase, the Entecavir enantiomter can be separated during 30 ℃ of column temperatures.Empirical tests, this method can still because contain the modifier trifluoroacetic acid in the moving phase, cause baseline wander with the Entecavir stage enantiomer separation, can't accurately measure the content of enantiomter in the finished product, can not effectively control the quality of Entecavir raw material; Simultaneously because the chiral chromatographic column stationary phase has memory effect to modifier, be explicitly shown as on the chromatographic column operation instructions and guarantee the functional of chromatographic column, after using modifier, need to adopt absolute ethyl alcohols (chromatographic grade) to re-use the normal developing method after 3 hours at every turn and preserve, so repeatedly not only complex operation but also serviceable life of damaging pillar repeatedly with the flow velocity flushing of 0.5ml/min.
Through repetition test, the inventor finds, with amylose-three (3,5-3,5-dimethylphenyl-carbamate) is the chiral chromatographic column of filler, with normal hexane-low-alcohol solution is moving phase, and need not to add the acid modification agent can effectively separate the Entecavir chiral isomer, and baseline is steady under this method, the main peak peak shape is better, helps quantitative test; Because this condition is gentle (not containing modifier) comparatively, chromatographic column directly adopts conventional purging method to get final product after using, the cost that saves time, and the while can guarantee that chromatographic column has relatively long serviceable life.
Summary of the invention
The object of the present invention is to provide the efficient liquid-phase chromatography method of a kind of separation determination Entecavir and chiral isomer thereof, thereby realize the separation and the mensuration of Entecavir chiral isomer, control the product quality of Entecavir effectively.
For realizing purpose of the present invention, in one embodiment, method with liquid chromatography for separating and determining Entecavir and optical isomer thereof of the present invention, it is characterized in that: with three (3, the 5-3,5-dimethylphenyl)-the carbamate amylose is the chiral chromatographic column of filler, is moving phase with normal hexane-low-alcohol solution.Here said liquid-phase chromatography method comprises high performance liquid chromatography or high performance liquid chromatography-mass spectroscopy coupling.
Above-mentioned said be that the chiral chromatographic column of filler is selected from the chromatographic column that the trade mark is Daicel CHIRALPAK AD, Daicel CHIRALPAK AD-H and Daicel CHIRALPAK IC with three (3, the 5-3,5-dimethylphenyl)-carbamate amyloses.
Above-mentioned said lower alcohol is selected from following compound: methyl alcohol, absolute ethyl alcohol, propyl alcohol, isopropyl alcohol are preferably absolute ethyl alcohol or isopropyl alcohol.
The method of the invention described above, the volume ratio of its moving phase normal hexane-lower alcohol are 25: 75～75: 25., and preferred volume ratio is 60: 40, and more preferably the volume ratio of normal hexane-absolute ethyl alcohol or isopropyl alcohol is 60: 40.
The described method with liquid chromatography for separating and determining Entecavir and optical isomer thereof of the invention described above also further may further comprise the steps:
(1) it is an amount of to take by weighing the Entecavir sample, uses the anhydrous alcohol solution sample, is mixed with the sample solution that every 1ml contains Entecavir 0.1mg～5mg;
(2) flow rate of mobile phase being set is 0.5～2.0ml/min, and the flow velocity of moving phase is preferably 1.3ml/min; The detection wavelength is 205nm～260nm, and the preferred detection wavelength is 254nm, and the column oven temperature of chromatographic column is 25 ℃～45 ℃, and optimum column temperature oven temperature, degree is 40 ℃;
(3) get sample solution 2～50 μ l injecting chromatographs of (1), finish the separation determination of Entecavir and optical isomer.
Wherein:
The model of high performance liquid chromatograph has no special requirements, and the chromatograph that the present invention adopts is Agilent 1100 type high performance liquid chromatographs.
Chromatographic column: preferred Daicel CHIRALPAK AD chiral column (250mm * 4.6mm)
Column temperature: preferred 40 ℃
Moving phase: preferred normal hexane-absolute ethyl alcohol (60: 40)
Sample size: 10 μ l
In another embodiment, method with liquid chromatography for separating and determining Entecavir and optical isomer thereof of the present invention of the present invention, it is characterized in that: with three (3, the 5-3,5-dimethylphenyl)-the carbamate amylose is the chiral chromatographic column of filler, with normal hexane-low-alcohol solution is moving phase, its volume ratio is 25: 75～75: 25, and its method may further comprise the steps:
(1) it is an amount of to take by weighing the Entecavir sample, with lower alcohol (as absolute ethyl alcohol) sample dissolution, is mixed with the sample solution that every 1ml contains Entecavir 0.1mg～5mg;
(2) flow rate of mobile phase being set is 0.5～2.0ml/min, and the flow velocity of moving phase is preferably 1.3ml/min; The detection wavelength is 205nm～260nm, and the preferred detection wavelength is 254nm, and the column oven temperature of chromatographic column is 25 ℃～45 ℃, and optimum column temperature oven temperature, degree is 40 ℃;
(3) get sample solution 2～50 μ l of (1), preferred 10 μ l injecting chromatographs are finished the separation determination of Entecavir and optical isomer.
Wherein:
The model of high performance liquid chromatograph has no special requirements, and the chromatograph that the present invention adopts is Agilent 1100 type high performance liquid chromatographs.
In above-mentioned specific embodiments, three (3, the 5-3,5-dimethylphenyl)-the carbamate amylose is that the chiral chromatographic column of filler is selected from the chromatographic column that the trade mark is Daicel CHIRALPAK AD, Daicel CHIRALPAK AD-H and Daicel CHIRALPAKIC, preferred Daicel CHIRALPAKAD; Lower alcohol is selected from following compound: methyl alcohol, absolute ethyl alcohol, propyl alcohol, isopropyl alcohol are preferably absolute ethyl alcohol or isopropyl alcohol, more preferably absolute ethyl alcohol; The volume ratio of its moving phase normal hexane-lower alcohol is 25: 75～75: 25., and preferred volume ratio is 60: 40, and more preferably the volume ratio of normal hexane-absolute ethyl alcohol or isopropyl alcohol is 60: 40.
In above-mentioned specific embodiments, the lower alcohol in the step (1) is preferably identical with the lower alcohol of moving phase, preferred absolute ethyl alcohol.
The present invention adopts CHIRALPAK AD chiral chromatographic column, the effectively optical isomer of separation determination Entecavir; Selecting absolute ethyl alcohol for use is the dissolution with solvents sample, has guaranteed the stability of solution; Selecting sampling volume is 10 μ l, and column temperature is 40 ℃, has improved the symmetry of chromatographic peak.The invention solves the quantitative measurement problem of the optical isomer of separation determination Entecavir, thereby effectively guaranteed the quality controllable of Entecavir.
Description of drawings:
Fig. 1: the high-efficient liquid phase chromatogram of blank solvent
Fig. 2: the high-efficient liquid phase chromatogram of Entecavir raw material and Entecavir enantiomerism mixed liquor
Fig. 3: the high-efficient liquid phase chromatogram of Entecavir raw material
Fig. 4: the high-efficient liquid phase chromatogram of auxiliary material blank
Fig. 5: the high-efficient liquid phase chromatogram of Entecavir sheet
Embodiment:
Embodiment 1
Instrument and condition
High performance liquid chromatograph: Agilent 1100 type high performance liquid chromatographs
Chromatographic column: Daicel CHIRALPAK AD chiral column (250mm * 4.6mm)
Column temperature: 40 ℃
Moving phase: normal hexane-absolute ethyl alcohol (60: 40)
Flow velocity: 1.3ml/min
Detect wavelength: 254nm
Sample size: 10 μ l
Implementation step
Get Entecavir raw material 25mg and Entecavir enantiomter 12.5mg respectively, put in the same 50ml measuring bottle, add anhydrous alcohol solution and be diluted to scale, shake up, as need testing solution.
Get blank reagent solution and need testing solution respectively, carry out efficient liquid phase chromatographic analysis by above-mentioned condition, the record chromatogram the results are shown in Figure 1 and Fig. 2.
Retention time is that 3.807 minutes chromatographic peak is the chromatographic peak of Entecavir enantiomter among Fig. 2; 5.963 minute chromatographic peak be the chromatographic peak of Entecavir, as seen from the figure, Entecavir and its enantiomter can reach the baseline separation degree.
Instrument and condition
High performance liquid chromatograph: Agilent 1100 type high performance liquid chromatographs
Chromatographic column: Daicel CHIRALPAK AD chiral column (250mm * 4.6mm)
Column temperature: 40 ℃
Moving phase: normal hexane-absolute ethyl alcohol (60: 40)
Flow velocity: 1.3ml/min
Detect wavelength: 254nm
Sample size: 10 μ l
Implementation step
Get Entecavir raw material 25mg, put in the 50ml measuring bottle, add anhydrous alcohol solution and be diluted to scale, shake up, as need testing solution.
Get need testing solution, carry out efficient liquid phase chromatographic analysis by above-mentioned condition, the record chromatogram the results are shown in Figure 3.
Fig. 3 proves that the optical purity of Entecavir reaches the bulk drug requirement, and this law can be used for the quality monitoring of Entecavir.
Embodiment 3
Instrument and condition
High performance liquid chromatograph: Agilent 1100 type high performance liquid chromatographs
Chromatographic column: Daicel CHIRALPAK AD chiral column (250mm * 4.6mm)
Column temperature: 40 ℃
Moving phase: normal hexane-absolute ethyl alcohol (60: 40)
Flow velocity: 1.3ml/min
Detect wavelength: 254nm
Sample size: 10 μ l
Implementation step
It is an amount of to get the Entecavir sheet, is equivalent to Entecavir 12.5mg approximately, puts in the 25ml measuring bottle, add the absolute ethyl alcohol sonicated and make dissolving, and with ethanol dilution to scale, shake up, filter, get subsequent filtrate as need testing solution.
Get need testing solution, carry out efficient liquid phase chromatographic analysis by above-mentioned condition, record chromatogram, and carry out the test of blank auxiliary material with method the results are shown in Figure 4, Fig. 5.
Fig. 4 proves that blank auxiliary material does not disturb the mensuration of this product, and Fig. 5 proves that this law can be used for the quality monitoring of Entecavir sheet.
Show from Fig. 1-Fig. 4: method of the present invention, can clearly Entecavir be separated with its chiral isomer, drift about, help quantitative Analysis with base line, can calculate the content of chiral isomer exactly, from the product quality of effective control Entecavir.
Claims (8)
1. method that adopts high performance liquid chromatography (HPLC) separation determination Entecavir and enantiomter thereof, it is characterized in that adopting three (3, the 5-3,5-dimethylphenyl)-the carbamate amylose is the chiral chromatographic column of filler, with normal hexane-low-alcohol solution is moving phase, and normal hexane-low-alcohol solution volume ratio is 25: 75～75: 25.
2. method according to claim 1 is characterized in that: described chiral chromatographic column is selected from the chromatographic column that the trade mark is Daicel CHIRALPAKAD, Daicel CHIRALPAK AD-H and Daicel CHIRALPAK IC.
3. method according to claim 1 is characterized in that described normal hexane-low-alcohol solution volume ratio is 60: 40.
4. according to claim 1 or 3 described methods, it is characterized in that described lower alcohol is selected from methyl alcohol, absolute ethyl alcohol, propyl alcohol and isopropyl alcohol.
5. method according to claim 4 is characterized in that described lower alcohol is absolute ethyl alcohol or isopropyl alcohol.
6. method according to claim 1 is characterized in that described method also further comprises following steps:
A) preparation of need testing solution: get Entecavir or contain the preparation of Entecavir an amount of, add the anhydrous alcohol solution sample, be mixed with every 1ml and contain the sample solution of Entecavir 0.1mg to 5mg as need testing solution;
B) flow velocity that moving phase is set is 0.5～2.0ml/min, and being provided with and detecting wavelength is 205nm to 260nm, and the column temperature that chromatographic column is set is 25 ℃ to 45 ℃;
C) getting a) sample solution, sample size is set is 5 μ l～100 μ l, injects liquid chromatograph, and the record chromatogram is finished the separation determination of Entecavir and optical isomer.
7. method according to claim 6, the column temperature of described step b) are 40 ℃.
8. method according to claim 6, the detection wavelength of described step b) is 254nm.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102260262A (en) * | 2011-05-31 | 2011-11-30 | 刘全胜 | Method for purifying entecavir and tablets |
| CN102305837A (en) * | 2011-05-21 | 2012-01-04 | 江苏诺泰制药技术有限公司 | Detection method for controlling content of entecavir, and related intermediate substance and isomer thereof |
| CN103675185A (en) * | 2013-12-10 | 2014-03-26 | 上海景峰制药股份有限公司 | Method for determining all-trans isomers in entecavir tablets by high performance liquid chromatography |
| CN104634887A (en) * | 2013-11-11 | 2015-05-20 | 广东东阳光药业有限公司 | Method for separating and measuring ticagrelor and optical isomer of ticagrelor |
| CN105675733A (en) * | 2014-11-17 | 2016-06-15 | 重庆医药工业研究院有限责任公司 | Method for using liquid chromatography method for separation and determination of trelagliptin succinate and trelagliptin succinate optical isomers |
| CN109239215A (en) * | 2018-09-20 | 2019-01-18 | 杭州佰辰医学检验所有限公司 | The method of Entecavir in isotopic dilution ultra performance liquid chromatography mass spectrometry serum or blood plasma |
| WO2020010740A1 (en) * | 2018-07-09 | 2020-01-16 | 深圳翰宇药业股份有限公司 | Detection method for vildagliptin enantiomer |
| CN115290800A (en) * | 2022-10-08 | 2022-11-04 | 江西省药品检验检测研究院 | Method for splitting antofloxacin enantiomer by chiral stationary phase method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102305837A (en) * | 2011-05-21 | 2012-01-04 | 江苏诺泰制药技术有限公司 | Detection method for controlling content of entecavir, and related intermediate substance and isomer thereof |
| CN102260262A (en) * | 2011-05-31 | 2011-11-30 | 刘全胜 | Method for purifying entecavir and tablets |
| CN102260262B (en) * | 2011-05-31 | 2012-12-19 | 刘全胜 | Method for purifying entecavir and tablets |
| CN104634887A (en) * | 2013-11-11 | 2015-05-20 | 广东东阳光药业有限公司 | Method for separating and measuring ticagrelor and optical isomer of ticagrelor |
| CN103675185A (en) * | 2013-12-10 | 2014-03-26 | 上海景峰制药股份有限公司 | Method for determining all-trans isomers in entecavir tablets by high performance liquid chromatography |
| CN103675185B (en) * | 2013-12-10 | 2015-10-07 | 上海景峰制药股份有限公司 | A kind of method of all trans isomer in high effective liquid chromatography for measuring Entecavir tablet |
| CN105675733A (en) * | 2014-11-17 | 2016-06-15 | 重庆医药工业研究院有限责任公司 | Method for using liquid chromatography method for separation and determination of trelagliptin succinate and trelagliptin succinate optical isomers |
| CN105675733B (en) * | 2014-11-17 | 2020-01-31 | 重庆医药工业研究院有限责任公司 | method for separating and measuring trelagliptin succinate and optical isomers thereof by liquid chromatography |
| WO2020010740A1 (en) * | 2018-07-09 | 2020-01-16 | 深圳翰宇药业股份有限公司 | Detection method for vildagliptin enantiomer |
| CN109239215A (en) * | 2018-09-20 | 2019-01-18 | 杭州佰辰医学检验所有限公司 | The method of Entecavir in isotopic dilution ultra performance liquid chromatography mass spectrometry serum or blood plasma |
| CN115290800A (en) * | 2022-10-08 | 2022-11-04 | 江西省药品检验检测研究院 | Method for splitting antofloxacin enantiomer by chiral stationary phase method |
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Application publication date: 20100505 |