CN110057959A - A kind of analysis method of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance - Google Patents
A kind of analysis method of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance Download PDFInfo
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- CN110057959A CN110057959A CN201910438360.2A CN201910438360A CN110057959A CN 110057959 A CN110057959 A CN 110057959A CN 201910438360 A CN201910438360 A CN 201910438360A CN 110057959 A CN110057959 A CN 110057959A
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- G—PHYSICS
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The present invention provides a kind of high effective liquid chromatography for measuring Febustat intermediate 2- (3- formoxyl -4- hydroxy phenyl) analysis method of -4- methyl-5-thiazole formic acid ethyl ester in relation to substance; the analysis of principal component Self-control method, chromatographic condition are as follows: chromatographic column Agilent ZORBAX C18 are carried out using RP-HPLC；Flow velocity 0.9-1.1ml/min；Detection wavelength 315nm；20-25 DEG C of column temperature；Sample volume: 20 μ l；Mobile phase A: 0.1% (v/v) trifluoroacetic acid, B: acetonitrile carries out gradient elution；This method is easy to produce and quality control process in product quality control, the extent of reaction of subsequent reactions and the quality of final products can be improved, there is at low cost, simple and easy, accuracy and precision is high, stability and the advantages of favorable reproducibility, high sensitivity.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical fields, in particular to a kind of high effective liquid chromatography for measuring Fei Busi
His intermediate 2- (3- formoxyl -4- hydroxy phenyl) analysis method of -4- methyl-5-thiazole formic acid ethyl ester in relation to substance.
Background technique
Febustat (Febuxostat), entitled 2- [(3- cyano-4-isobutoxy) the phenyl] -4- methyl -5- of chemistry
Thiazole carboxylic acid, molecular formula: C16H16N2O3S, molecular weight: 316.37.
Its structural formula are as follows:
Febustat is novel non-purines XOR inhibitor, has high selectivity to XOR, and to oxidized form and also
The XOR of prototype all has significant inhibiting effect, and clinically for treating the excessively high disease of uric acid (gout), effect is better than not fast
Alcohol, safe and effective to light moderate hepatic and kidney function obstacle person, Small side effects, China's patient with gout is numerous, and XOR inhibitor is preferred use
Medicine, therefore welcome by patient.
The common synthetic route for preparing Febustat is to be with 2- (4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester
Starting material, by aldehyde radicalization again with isobutyl bromide, the chemical reactive synthesis by 4 steps such as isobutyl, cyanalation, hydrolysis is non-
Bu Sita.Currently, Febustat important intermediate 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester,
By raw material 2- (4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, through aldehyde radical, i.e. formylated process is obtained, need to be to reaction
It is controlled, raw material residual peak area in aldehyde radical produce product is made to be not greater than 0.6 times of contrast solution main peak area (0.6%),
Its single impurity peak area is not greater than 3 times of main peak area (3%) of control, and the sum of each impurity peak area is not greater than contrast solution
7 times (7%) of main peak peak area, to improve the quality of final products Febustat.
In prior art, high performance liquid chromatography is to measure one of the important measuring method of Febustat, such as:
A kind of measurement method of Febustat and its preparation in relation to substance of CN103163227A, realize Febustat and its
Inspection of the preparation in relation to substance.CN101881756B high-efficient liquid phase chromatogram technique analysis separates the side of Febustat and its intermediate
Method realizes the separation determination of Febustat and its intermediate impurities.In a kind of Febustat synthesis process of CN102565225A
Using the method for high effective liquid chromatography for measuring related substances, provide in Febustat final product to synthesis process intermediates
Detection.
It has been investigated that in the actual operation process, the above method has the disadvantage that
(1) Febustat intermediate 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid in the prior art
The purity of ethyl ester and the analysis method of related substances have no；
(2) between partial impurities and intermediate 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester
Polarity it is close, and the different polarities of partial impurities are larger, and intermediate 2- (3- formyl cannot be effectively detected in real work
Base -4- hydroxy phenyl) in -4- methyl-5-thiazole formic acid ethyl ester sample respectively at swarming.
Summary of the invention
The object of the present invention is to provide a kind of high effective liquid chromatography for measuring Febustat intermediate 2- (3- formyls
Base -4- hydroxy phenyl) the analysis method of -4- methyl-5-thiazole formic acid ethyl ester in relation to substance, reach main peak with other impurities peak
Baseline separation carries out comprehensive and stringent control to reaction impurities, and provides integrity authentication scheme, easy to operate, precision
Height, stability is good, favorable reproducibility.
The present invention provides a kind of high effective liquid chromatography for measuring Febustat intermediate 2- (3- formoxyl -4- hydroxy benzenes
Base) the analysis method of -4- methyl-5-thiazole formic acid ethyl ester in relation to substance, it is characterised in that: including following operating procedure:
(1) solution is prepared: being taken 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, is added acetonitrile-water
(1:1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.2mg, as test solution；Precision measures test sample
Solution 1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution；
(2) contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records color
For spectrogram to 4 times of principal component peak retention time, chromatographic condition is as follows:
Chromatographic column: octadecylsilane chemically bonded silica is filler；
Detection wavelength: 315nm；
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
(3) measuring method: content is calculated using by principal component Self-control method.
Described Febustat intermediate 2- (3- formoxyl -4- the hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester is related
Substance be 2- (4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, other single impurity and each impurity peak area and.
A kind of described high effective liquid chromatography for measuring Febustat intermediate 2- (3- formoxyl -4- the hydroxy phenyl) -4-
Analysis method of the methyl-5-thiazole formic acid ethyl ester in relation to substance, it is characterised in that: column size described in step (2) is 5
μm C18250*4.6mm。
A kind of described high effective liquid chromatography for measuring Febustat intermediate 2- (3- formoxyl -4- the hydroxy phenyl) -4-
Analysis method of the methyl-5-thiazole formic acid ethyl ester in relation to substance, it is characterised in that: the flow velocity of mobile phase is 0.9- in step (2)
1.1ml/min。
A kind of described high effective liquid chromatography for measuring Febustat intermediate 2- (3- formoxyl -4- the hydroxy phenyl) -4-
Analysis method of the methyl-5-thiazole formic acid ethyl ester in relation to substance, it is characterised in that: column temperature is 20-25 DEG C in step (2).
A kind of described high effective liquid chromatography for measuring Febustat intermediate 2- (3- formoxyl -4- the hydroxy phenyl) -4-
Analysis method of the methyl-5-thiazole formic acid ethyl ester in relation to substance, it is characterised in that: the 2- (3- formoxyl -4- hydroxyl in step (2)
Base phenyl) -4- methyl-5-thiazole formic acid ethyl ester and the adjacent separating degree in relation to substance should be greater than 1.5.
A kind of described high effective liquid chromatography for measuring Febustat intermediate 2- (3- formoxyl -4- the hydroxy phenyl) -4-
Analysis method of the methyl-5-thiazole formic acid ethyl ester in relation to substance, it is characterised in that: the test solution chromatogram in step (3)
In if any with the consistent chromatographic peak of 2- (4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester peak retention time, peak area is not
It obtains and is greater than 0.6 times of contrast solution main peak area (0.6%), other single impurity peak areas are not greater than 3 times of main peak area of control
(3%), the sum of each impurity peak area is not greater than 7 times (7%) of contrast solution main peak peak area.
Particularly, the flow visualizing of this detection method and gradient elution ratio be the time of the invention, place, environment and
It is optimal under operator's limitation, and flow visualizing without being limited thereto and gradient elution ratio, ratio, concentration to secondary flow visualizing
And gradient elution program does suitably modified, adjustment and equivalent replacement, without departing from technical idea and protection scope of the invention.
The advantages of this law, is: can preferably make each chromatographic peak separation in sample and appearance is most, can effectively measure non-
Correlation involved in Bu Sita synthesis process intermediate 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester
Substance, to improve the quality of final products Febustat.It is wide with the scope of application, step is simple, analysis method it is time-consuming it is short, analysis
It is at low cost；The advantages that analysis method is high to analysis result accuracy, the range of linearity is wide, favorable reproducibility, analysis condition are stablized, can be wide
The general on-line analysis being applied in production.
Detailed description of the invention
Fig. 1: the contrast solution high-efficient liquid phase chromatogram separated according to 1 condition of embodiment；
Fig. 2: the test solution high-efficient liquid phase chromatogram separated according to 1 condition of embodiment；
Fig. 3: the contrast solution high-efficient liquid phase chromatogram separated according to 2 condition of embodiment；
Fig. 4: the test solution high-efficient liquid phase chromatogram separated according to 2 condition of embodiment；
Fig. 5: the contrast solution high-efficient liquid phase chromatogram separated according to 3 condition of embodiment；
Fig. 6: the test solution high-efficient liquid phase chromatogram separated according to 3 condition of embodiment；
Fig. 7: the contrast solution high-efficient liquid phase chromatogram separated according to 4 condition of embodiment；
Fig. 8: the test solution high-efficient liquid phase chromatogram separated according to 4 condition of embodiment；
Fig. 9: the contrast solution high-efficient liquid phase chromatogram separated according to 5 condition of embodiment；
Figure 10: the test solution high-efficient liquid phase chromatogram separated according to 5 condition of embodiment；
Figure 11: the contrast solution high-efficient liquid phase chromatogram separated according to 6 condition of embodiment；
Figure 12: the test solution high-efficient liquid phase chromatogram separated according to 6 condition of embodiment；
Figure 13: the contrast solution high-efficient liquid phase chromatogram separated according to 7 condition of embodiment；
Figure 14: the test solution high-efficient liquid phase chromatogram separated according to 7 condition of embodiment.
Specific embodiment
Form makees further supplementary explanation to above content by the following examples, but should not be construed the present invention and only limit
In following instance.The techniques implemented on the basis of the foregoing are all within the scope of the present invention.
Embodiment 1
Experiment equipment and reagent
(1) laboratory apparatus
Instrument: Shimadzu liquid chromatograph LC-20AT matches UV detector
(2) chromatographic condition
Chromatographic column: Agilent ZORBAX SB-C185.0 μm C18250*4.6mm
Flow velocity: 1.0ml/min
Detection wavelength: 315nm
Column temperature: 25 DEG C
Sample volume: 20 μ L
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
| Time (minute) | Mobile phase A (%) (v/v) | Mobile phase B (%) (v/v) |
| 0 | 40 | 60 |
| 10 | 40 | 60 |
| 25 | 10 | 90 |
| 35 | 10 | 90 |
| 40 | 40 | 60 |
(1) experiment reagent
Acetonitrile: Beijing lark prestige Science and Technology Ltd., HPLC grades
Trifluoroacetic acid: TEDIA is analyzed pure
Implementation steps:
Solution prepare: take 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, add acetonitrile-water (1:
1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.2mg, as test solution；Precision measures test solution
1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution.
Contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records chromatogram
To 4 times of principal component peak retention time.The results are shown in Table shown in 1, table 2 and attached drawing 1, attached drawing 2, between each impurity with 2- (3- formyl
Base -4- hydroxy phenyl) separating degree between -4- methyl-5-thiazole formic acid ethyl ester is greater than 1.5, show that this method specificity is good.
1 separating degree result of study of table
| Solution | Separating degree |
| Control | 8.58 |
| Test sample | 12.8 |
2 principal component Self-control method result of study of table
Embodiment 2
Experiment equipment and reagent
(1) laboratory apparatus
Instrument: Shimadzu liquid chromatograph LC-20AT matches UV detector
(2) chromatographic condition
Chromatographic column: Agilent ZORBAX SB-C185.0 μm C18250*4.6mm
Flow velocity: 1.1ml/min
Detection wavelength: 315nm
Column temperature: 25 DEG C
Sample volume: 20 μ L
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
| Time (minute) | Mobile phase A (%) (v/v) | Mobile phase B (%) (v/v) |
| 0 | 40 | 60 |
| 10 | 40 | 60 |
| 25 | 10 | 90 |
| 35 | 10 | 90 |
| 40 | 40 | 60 |
(1) experiment reagent
Acetonitrile: Beijing lark prestige Science and Technology Ltd., HPLC grades
Trifluoroacetic acid: TEDIA is analyzed pure
Implementation steps:
Solution prepare: take 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, add acetonitrile-water (1:
1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.2mg, as test solution；Precision measures test solution
1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution.
Contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records chromatogram
To 4 times of principal component peak retention time.It the results are shown in Table shown in 3, table 4 and attached drawing 3, attached drawing 4, and, show we between each impurity
Method specificity is good.Between each impurity between 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester
Separating degree is greater than 1.5, shows that this method specificity is good.
3 separating degree result of study of table
| Solution | Separating degree |
| Control | 15.9 |
| Test sample | 8.78 |
4 principal component Self-control method result of study of table
Embodiment 3
Experiment equipment and reagent
(1) laboratory apparatus
Instrument: Shimadzu liquid chromatograph LC-20AT matches UV detector
(2) chromatographic condition
Chromatographic column: Agilent ZORBAX SB-C185.0 μm C18250*4.6mm
Flow velocity: 0.9ml/min
Detection wavelength: 315nm
Column temperature: 25 DEG C
Sample volume: 20 μ L
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
| Time (minute) | Mobile phase A (%) (v/v) | Mobile phase B (%) (v/v) |
| 0 | 40 | 60 |
| 10 | 40 | 60 |
| 25 | 10 | 90 |
| 35 | 10 | 90 |
| 40 | 40 | 60 |
(1) experiment reagent
Acetonitrile: Beijing lark prestige Science and Technology Ltd., HPLC grades
Trifluoroacetic acid: TEDIA is analyzed pure
Implementation steps:
Solution prepare: take 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, add acetonitrile-water (1:
1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.2mg, as test solution；Precision measures test solution
1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution.
Contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records chromatogram
To 4 times of principal component peak retention time.The results are shown in Table shown in 5, table 6 and attached drawing 5, attached drawing 6, between each impurity with 2- (3- formyl
Base -4- hydroxy phenyl) separating degree between -4- methyl-5-thiazole formic acid ethyl ester is greater than 1.5, show that this method specificity is good.
5 separating degree result of study of table
| Solution | Separating degree |
| Control | 2.84 |
| Test sample | 15.5 |
6 principal component Self-control method result of study of table
Embodiment 4
Experiment equipment and reagent
(1) laboratory apparatus
Instrument: Shimadzu liquid chromatograph LC-20AT matches UV detector
(2) chromatographic condition
Chromatographic column: Agilent ZORBAX SB-C185.0 μm C18250*4.6mm
Flow velocity: 1.0ml/min
Detection wavelength: 315nm
Column temperature: 20 DEG C
Sample volume: 20 μ L
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
| Time (minute) | Mobile phase A (%) (v/v) | Mobile phase B (%) (v/v) |
| 0 | 40 | 60 |
| 10 | 40 | 60 |
| 25 | 10 | 90 |
| 35 | 10 | 90 |
| 40 | 40 | 60 |
(1) experiment reagent
Acetonitrile: Beijing lark prestige Science and Technology Ltd., HPLC grades
Trifluoroacetic acid: TEDIA is analyzed pure
Implementation steps:
Solution prepare: take 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, add acetonitrile-water (1:
1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.2mg, as test solution；Precision measures test solution
1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution.
Contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records chromatogram
To 4 times of principal component peak retention time.The results are shown in Table shown in 7, table 8 and attached drawing 7, attached drawing 8, between each impurity with 2- (3- formyl
Base -4- hydroxy phenyl) separating degree between -4- methyl-5-thiazole formic acid ethyl ester is greater than 1.5, show that this method specificity is good.
7 separating degree result of study of table
| Solution | Separating degree |
| Control | 17.6 |
| Test sample | 9.01 |
8 principal component Self-control method result of study of table
Embodiment 5
Experiment equipment and reagent
(1) laboratory apparatus
Instrument: Shimadzu liquid chromatograph LC-20AT matches UV detector
(2) chromatographic condition
Chromatographic column: Agilent ZORBAX SB-C185.0 μm C18250*4.6mm
Flow velocity: 1.0ml/min
Detection wavelength: 315nm
Column temperature: 22 DEG C
Sample volume: 20 μ L
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
(1) experiment reagent
Acetonitrile: Beijing lark prestige Science and Technology Ltd., HPLC grades
Trifluoroacetic acid: TEDIA is analyzed pure
Implementation steps:
Solution prepare: take 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, add acetonitrile-water (1:
1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.2mg, as test solution；Precision measures test solution
1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution.
Contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records chromatogram
To 4 times of principal component peak retention time.The results are shown in Table shown in 9, table 10 and attached drawing 9, attached drawing 10, between each impurity with 2- (3- first
Acyl group -4- hydroxy phenyl) separating degree between -4- methyl-5-thiazole formic acid ethyl ester is greater than 1.5, show that this method specificity is good
It is good.
9 separating degree result of study of table
| Solution | Separating degree |
| Control | 17.5 |
| Test sample | 8.53 |
10 principal component Self-control method result of study of table
Embodiment 6
Experiment equipment and reagent
(1) laboratory apparatus
Instrument: Shimadzu liquid chromatograph LC-20AT matches UV detector
(2) chromatographic condition
Chromatographic column: Agilent ZORBAX SB-C185.0 μm C18250*4.6mm
Flow velocity: 1.0ml/min
Detection wavelength: 315nm
Column temperature: 25 DEG C
Sample volume: 20 μ L
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
| Time (minute) | Mobile phase A (%) (v/v) | Mobile phase B (%) (v/v) |
| 0 | 40 | 60 |
| 10 | 40 | 60 |
| 25 | 10 | 90 |
| 35 | 10 | 90 |
| 40 | 40 | 60 |
(1) experiment reagent
Acetonitrile: Beijing lark prestige Science and Technology Ltd., HPLC grades
Trifluoroacetic acid: TEDIA is analyzed pure
Implementation steps:
Solution prepare: take 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, add acetonitrile-water (1:
1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.1mg, as test solution；Precision measures test solution
1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution.
Contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records chromatogram
To 4 times of principal component peak retention time.The results are shown in Table shown in 11, table 12 and attached drawing 11, attached drawing 12, between each impurity with 2- (3-
Formoxyl -4- hydroxy phenyl) separating degree between -4- methyl-5-thiazole formic acid ethyl ester is greater than 1.5, show this method specificity
Well.
11 separating degree result of study of table
12 principal component Self-control method result of study of table
Embodiment 7
Experiment equipment and reagent
(1) laboratory apparatus
Instrument: Shimadzu liquid chromatograph LC-20AT matches UV detector
(2) chromatographic condition
Chromatographic column: Agilent ZORBAX SB-C185.0 μm C18250*4.6mm
Flow velocity: 1.0ml/min
Detection wavelength: 315nm
Column temperature: 25 DEG C
Sample volume: 20 μ L
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
| Time (minute) | Mobile phase A (%) (v/v) | Mobile phase B (%) (v/v) |
| 0 | 40 | 60 |
| 10 | 40 | 60 |
| 25 | 10 | 90 |
| 35 | 10 | 90 |
| 40 | 40 | 60 |
(1) experiment reagent
Acetonitrile: Beijing lark prestige Science and Technology Ltd., HPLC grades
Trifluoroacetic acid: TEDIA is analyzed pure
Implementation steps:
Solution prepare: take 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, add acetonitrile-water (1:
1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.3mg, as test solution；Precision measures test solution
1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution.
Contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records chromatogram
To 4 times of principal component peak retention time.The results are shown in Table shown in 13, table 14 and attached drawing 1, attached drawing 2, between each impurity with 2- (3- first
Acyl group -4- hydroxy phenyl) separating degree between -4- methyl-5-thiazole formic acid ethyl ester is greater than 1.5, show that this method specificity is good
It is good.
13 separating degree result of study of table
| Solution | Separating degree |
| Control | 17.7 |
| Test sample | 7.92 |
14 principal component Self-control method result of study of table
Claims (7)
1. a kind of analysis method of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance, it is characterised in that:
(1) solution is prepared:
Febustat intermediate 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester is taken, acetonitrile-water is added
(1:1) (v/v) dissolves and dilutes the solution being made in every 1ml containing about 0.2mg, as test solution；Precision measures test sample
Solution 1ml is set in 100ml measuring bottle, is added acetonitrile-water (1:1) (v/v) to be diluted to scale, is shaken up, as contrast solution.
(2) contrast solution, 20 μ l of test solution injection liquid chromatograph are taken, liquid chromatograph is injected separately into, records chromatogram
To 4 times of principal component peak retention time, chromatographic condition is as follows:
Chromatographic column: octadecylsilane chemically bonded silica is filler；
Detection wavelength: 315nm；
Mobile phase A: 0.1% (v/v) trifluoroacetic acid
Mobile phase B: acetonitrile, according to the form below carry out gradient elution
(3) measuring method: content is calculated using by principal component Self-control method.
Described Febustat intermediate 2- (3- formoxyl -4- hydroxy phenyl) the related substance of -4- methyl-5-thiazole formic acid ethyl ester
Be 2- (4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester, other single impurity and each impurity peak area and.
2. a kind of analysis side of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance according to claim 1
Method, it is characterised in that: column size described in step (2) is 5 μm of C18250*4.6mm
3. according to a kind of analysis side of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance described in claim 1
Method, it is characterised in that: the flow velocity of mobile phase is 0.9-1.1ml/min in step (2).
4. a kind of analysis side of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance according to claim 1
Method, it is characterised in that: column temperature is 20-25 DEG C in step (2).
5. a kind of analysis side of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance according to claim 1
Method, it is characterised in that: there is 2- (3- formoxyl -4- hydroxy phenyl) -4- methyl-5-thiazole formic acid ethyl ester with adjacent in step (2)
The separating degree for closing substance should be greater than 1.5.
6. a kind of analysis side of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance according to claim 1
The analysis method of method, it is characterised in that: in the test solution chromatogram in step (3) if any with 2- (4- hydroxy phenyl) -4-
The consistent chromatographic peak of methyl-5-thiazole formic acid ethyl ester peak retention time, peak area are not greater than contrast solution main peak area 0.6
Times (0.6%), other single impurity peak areas are not greater than 3 times of main peak area (3%) of control, each impurity peak area and must not
Greater than 7 times (7%) of contrast solution main peak peak area.
7. a kind of analysis side of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance according to claim 1
Method, it is characterised in that: the test solution concentration in step (1) is 0.1mg-0.3mg.
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| CN115356407B (en) * | 2022-07-04 | 2023-09-19 | 广东恒健制药有限公司 | Method for detecting suspected genotoxic impurities in febuxostat tablet |
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