CN107490630A - The analyzing detecting method of the hydroxyquinazoline of 7 fluorine 4 - Google Patents
The analyzing detecting method of the hydroxyquinazoline of 7 fluorine 4 Download PDFInfo
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- CN107490630A CN107490630A CN201610408865.0A CN201610408865A CN107490630A CN 107490630 A CN107490630 A CN 107490630A CN 201610408865 A CN201610408865 A CN 201610408865A CN 107490630 A CN107490630 A CN 107490630A
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- hydroxyquinazolines
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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
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The present invention relates to the analyzing detecting method of the hydroxyquinazoline of 7 fluorine 4, quality control for the hydroxyquinazoline of 7 fluorine 4, it is the chromatographic column (C18 using octadecylsilane chemically bonded silica as filler, 4.6 × 250mm, 3 μm), using 0.05~0.15% phosphate aqueous solution and acetonitrile as mobile phase, gradient elution, using Detection wavelength as 247nm, column temperature is 25~35 DEG C, carries out high-efficient liquid phase chromatogram technique analysis detection.The analyzing detecting method of the present invention effectively can separate the hydroxyquinazoline of 7 fluorine 4 and its impurity, and this method has separating degree and a high sensitivity, and repeatability and durability are good, and analysis time is short, simple to operate, it is as a result reliable and stable the advantages of.
Description
Technical field
The present invention relates to a kind of analysis of the fluoro- 4- hydroxyquinazolines of HPLC analytical method, especially 7- detection
Method.
Background technology
Afatinib is aniline Kui that isoxazoline compound, be a kind of irreversible EGFR-HER2 dual tyrosine kinases by
Body inhibitor, it irreversible can be combined with EGFR-HER2 EGFR-TKs, suppress its tyrosine kinase activity, and then block
Tumour cell signal transduction leading EGFR-HER2, suppress transfer and the propagation of tumour cell, promote the apoptosis of tumour cell.
The fluoro- 4- hydroxyquinazolines of 7- are one of important intermediates for synthesizing Afatinib, and its chemical formula is C8H5FN2O, knot
Structure formula is：
Not yet record the analyzing detecting method of the intermediate in document, but the analysis detection of the intermediate to reaction controlling and
Yield, which improves, quality that is important, while also directly affecting finished product Afatinib, so establishing a kind of stable
Effective analyzing detecting method carries out quality control to the intermediate and is very important.
The content of the invention
It is an object of the invention to provide a kind of analyzing detecting method of the fluoro- 4- hydroxyquinazolines of 7-, for the fluoro- 4- hydroxyls of 7-
The quality control of base quinazoline.
In order to realize the purpose of the present invention, inventor finally obtains following technical scheme by lot of experiments：
The analyzing detecting method of the fluoro- 4- hydroxyquinazolines of 7-, is the chromatogram using octadecylsilane chemically bonded silica as filler
Post, with 0.05~0.15% phosphate aqueous solution (volume ratio) for mobile phase A, using acetonitrile as Mobile phase B, carry out gradient elution, bag
Include following steps：
A, take the fluoro- 4- hydroxyquinazolines of 7- appropriate, solubilizer dissolving, be configured to every 1ml fluoro- 4- hydroxyquinazolines containing 7-
0.041~0.311mg sample solution；
B, it is 0.7~1.1ml/min to set flow rate of mobile phase, and Detection wavelength 247nm, column temperature is 25~35 DEG C；
C, take a μ l of sample solution 10 to inject liquid chromatograph, perform gradient elution program, complete the fluoro- 4- hydroxyls quinolines of 7-
The analysis detection of oxazoline；
The specification of the chromatographic column is C18,4.6 × 250mm, 3 μm.
With volume basis, described gradient elution sets as follows：
Further, the setting of gradient elution is preferably：
Described mobile phase A is preferably 0.1% phosphate aqueous solution.
Described solvent is by 0.1% phosphate aqueous solution：Acetonitrile by volume 90:10 compositions.
Described sample solution concentration is preferably 0.2mg/ml.
The flow velocity of the mobile phase is preferably 1.0ml/min, and column temperature is preferably 30 DEG C.
The method of the present invention is described in detail in embodiment, and method validation has been carried out to the method for the present invention,
As a result prove：Analyzing detecting method of the present invention, effectively the fluoro- 4- hydroxyquinazolines of 7- and its impurity can be separated,
And this method separating degree and high sensitivity, repeatability and durability are good, and analysis time is short, simple to operate, as a result reliable and stable,
So as to the quality control for the fluoro- 4- hydroxyquinazolines of 7-, effective guarantee is provided for the quality of final finished.
Brief description of the drawings
The fluoro- 4- hydroxyquinazolines HPLC collection of illustrative plates of 7- of Fig. 1 embodiments 1.
The fluoro- 4- hydroxyquinazolines HPLC collection of illustrative plates of 7- of Fig. 2 embodiments 2.
The fluoro- 4- hydroxyquinazolines HPLC collection of illustrative plates of 7- of Fig. 3 embodiments 3.
The fluoro- 4- hydroxyquinazolines linear work curves of 7- of Fig. 4 embodiments 8.
Embodiment
It is the specific embodiment of the present invention below, technical scheme is further described, but it is of the invention
Protection domain be not limited to these embodiments.It is every to be included in this hair without departing substantially from the change of present inventive concept or equivalent substitute
Within bright protection domain.
Embodiment 1
Instrument and condition：The liquid chromatographic systems of Agilent 1260, chromatographic column：welch Ultimate XB-C18(4.6
× 250mm, 3 μm), Detection wavelength 247nm, 30 DEG C, flow velocity 1.0ml/min of column temperature, it is with 0.1% phosphate aqueous solution (volume ratio)
Mobile phase A, using acetonitrile as Mobile phase B, with volume basis, gradient elution is arranged to：
Experimental procedure：By the fluoro- 4- hydroxyquinazolines volume ratios 90 of 7-:10 0.1% phosphate aqueous solution and acetonitrile dissolving
And the solution that the fluoro- 4- hydroxyquinazolines 0.2mg containing 7- in every 1ml is made in dilution is quantified, as need testing solution, precision measures confession
The μ l of test sample solution 10 inject liquid chromatograph, carry out efficient liquid phase chromatographic analysis by above-mentioned condition, record chromatogram, as a result see attached
Fig. 1.
Accompanying drawing 1 shows, under the chromatographic condition, the fluoro- 4- hydroxyquinazolines peaks of 7- and impurity peaks can be kept completely separate, and peak
Shape is preferable, and separating degree is higher, and the retention time at the fluoro- 4- hydroxyquinazolines peaks of 7- is in 13.839min, separating degree 3.55.
Embodiment 2
Instrument and condition：The liquid chromatographic systems of Agilent 1260, chromatographic column：welch Ultimate XB-C18(4.6
× 250mm, 3 μm), Detection wavelength 247nm, 30 DEG C, flow velocity 0.7ml/min of column temperature, with 0.05% phosphate aqueous solution (volume ratio)
For mobile phase A, using acetonitrile as Mobile phase B, with volume basis, gradient elution is arranged to：
Experimental procedure：By the fluoro- 4- hydroxyquinazolines volume ratios 90 of 7-:10 0.1% phosphate aqueous solution and acetonitrile dissolving
And the solution that the fluoro- 4- hydroxyquinazolines 0.2mg containing 7- in every 1ml is made in dilution is quantified, as need testing solution, precision measures confession
The μ l of test sample solution 10 inject liquid chromatograph, carry out efficient liquid phase chromatographic analysis by above-mentioned condition, record chromatogram, as a result see attached
Fig. 2.
Accompanying drawing 2 shows, under the chromatographic condition, the fluoro- 4- hydroxyquinazolines peaks of 7- and impurity peaks can be kept completely separate, and peak
Shape is preferable, and separating degree is higher, and the retention time at the fluoro- 4- hydroxyquinazolines peaks of 7- is in 14.389min, separating degree 2.87.
Embodiment 3
Instrument and condition：The liquid chromatographic systems of Agilent 1260, chromatographic column：welch Ultimate XB-C18(4.6
× 250mm, 3 μm), Detection wavelength 247nm, 30 DEG C, flow velocity 1.1ml/min of column temperature, with 0.15% phosphate aqueous solution (volume ratio)
For mobile phase A, using acetonitrile as Mobile phase B, with volume basis, gradient elution is arranged to：
Experimental procedure：By the fluoro- 4- hydroxyquinazolines volume ratios 90 of 7-:10 0.1% phosphate aqueous solution and acetonitrile dissolving
And the solution that the fluoro- 4- hydroxyquinazolines 0.2mg containing 7- in every 1ml is made in dilution is quantified, as need testing solution, precision measures confession
The μ l of test sample solution 10 inject liquid chromatograph, carry out efficient liquid phase chromatographic analysis by above-mentioned condition, record chromatogram, as a result see attached
Fig. 3.
Accompanying drawing 3 shows, under the chromatographic condition, the fluoro- 4- hydroxyquinazolines peaks of 7- and impurity peaks can be kept completely separate, and peak
Shape is preferable, and separating degree is higher, and the retention time at the fluoro- 4- hydroxyquinazolines peaks of 7- is in 12.995min, separating degree 3.53.
Embodiment 4
System suitability is tested
Instrument and condition：With embodiment 1.
Experimental procedure：Take this product appropriate, it is accurately weighed, with volume ratio 90:10 0.1% phosphate aqueous solution and acetonitrile dissolving
And dilute and the solution containing 0.2mg in every 1ml is made, as need testing solution.Take need testing solution, continuous sample introduction six times, difference
The relative standard deviation of the fluoro- 4- hydroxyquinazolines peak peak areas of 7- and retention time is calculated, experimental result is shown in Table 1.
The fluoro- 4- hydroxyquinazolines system suitability experimental results of the 7- of table 1
As shown in Table 1, the symmetrical factor at the fluoro- 4- hydroxyquinazolines peaks of 7- is respectively less than 1.5, and number of theoretical plate is above 3000,
The relative standard deviation of peak area is 0.073% (limit 2.0%), and the relative standard deviation of retention time is 0.142% (limit
1.0%).It can be seen that under the chromatographic condition, the fluoro- 4- hydroxyquinazolines peak peak shapes of 7- are preferable, and relative standard deviation is smaller, institute
It is reliable and stable to obtain result.
Embodiment 5
Repeated experiment
Instrument and condition：With embodiment 1.
Experimental procedure：Take this product appropriate, it is accurately weighed, with volume ratio 90:10 0.1% phosphate aqueous solution and acetonitrile dissolving
And dilute and the solution containing 0.2mg in every 1ml is made, as need testing solution, 6 parts of need testing solutions are prepared with method.Take test sample
Solution, determined according to this method, calculate the fluoro- 4- hydroxyquinazolines contents of 7- by area normalization method, and it is inclined to calculate its relative standard
Difference, experimental result are shown in Table 2.
The fluoro- 4- hydroxyquinazolines repeated experiment results of the 7- of table 2
As shown in Table 2, the content of the fluoro- 4- hydroxyquinazolines of 7- does not have notable difference, relative standard in each need testing solution
Deviation is 0.001%, it is seen that the repeatability of this analyzing detecting method is good.
Embodiment 6
Durability is tested
Instrument and condition：The liquid chromatographic systems of Agilent 1260, chromatographic column：welch Ultimate XB-C18(4.6
× 250mm, 3 μm), Detection wavelength 247nm, mobile phase is the same as embodiment 1.
Experimental procedure：Take this product appropriate, it is accurately weighed, with volume ratio 90:10 0.1% phosphate aqueous solution and acetonitrile dissolving
And dilute and the solution containing 0.2mg in every 1ml is made, as need testing solution.Respectively by changing column temperature, flow velocity and pillar batch
It is secondary, the situation of change (being calculated by area normalization method) of the fluoro- 4- hydroxyquinazolines contents of 7- is recorded, experimental result is shown in Table 3.
The fluoro- 4- hydroxyquinazolines durability experimental results of the 7- of table 3
As shown in Table 3, after changing column temperature, flow velocity and pillar batch, the measurement result of the fluoro- 4- hydroxyquinazolines contents of 7- does not have
There is notable difference, it is seen that the good tolerance of analyzing detecting method of the present invention.
Embodiment 7
Test limit
Instrument and condition：With embodiment 1.
The fluoro- 4- hydroxyquinazolines about 10mg of 7- are taken, it is accurately weighed, put and storing solution is used as in 50ml measuring bottles.Add volume ratio 90:
10 0.1% phosphate aqueous solution and acetonitrile dissolving, using progressively dilution method, using concentration during S/N ≈ 3 as test limit concentration,
Now the concentration of the fluoro- 4- hydroxyquinazolines of 7- is 0.01773 μ g/ml, and detection is limited to 0.1773ng.It can be seen that this method and instrument
Sensitivity is higher.
Embodiment 8
Linearity and range
Instrument and condition：With embodiment 1.
Experimental procedure：The fluoro- 4- hydroxyquinazolines 41.4mg of 7- are taken, it is accurately weighed, put in 100ml measuring bottles, add volume ratio 90:
10 0.1% phosphate aqueous solution and acetonitrile dissolves and is diluted to scale, produces linear storing solution.Precision measures linear storing solution
1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, 6.0ml, 7.5ml are put in 10ml measuring bottles respectively, and solubilizer is diluted to scale, are shaken
It is even, determine in accordance with the law.Using the concentration of need testing solution as abscissa, entered using the fluoro- 4- hydroxyquinazolines peak peak areas of 7- as ordinate
Row linear regression, it is y=16673x+19.747 to obtain equation of linear regression, the results are shown in Table 4 and accompanying drawing 4.
The fluoro- 4- hydroxyquinazolines linear test results of the 7- of table 4
From table 4 and accompanying drawing 4, the coefficient R of equation of linear regression2=0.9999, it is seen that under the chromatographic condition,
The fluoro- 4- hydroxyquinazolines of 7- linear relationship in 0.041~0.311mg/ml concentration range is good.
Claims (5)
- The analyzing detecting method of the fluoro- 4- hydroxyquinazolines of 1.7-, carries out analysis detection, its feature exists using high performance liquid chromatography In comprising the following steps：A, take the fluoro- 4- hydroxyquinazolines of 7- appropriate, solubilizer dissolving, be configured to every 1ml fluoro- 4- hydroxyquinazolines 0.041 containing 7- ~0.311mg sample solution；B, it is 0.7~1.1ml/min to set flow rate of mobile phase, and Detection wavelength 247nm, column temperature is 25~35 DEG C；C, take the A μ l of sample solution 10 to inject liquid chromatograph, perform gradient elution program, complete the fluoro- 4- hydroxyquinazolines of 7- Analysis detection；Wherein, chromatographic column：C18,4.6 × 250mm, 3 μm；The mobile phase be with 0.05~0.15% phosphate aqueous solution (volume ratio) for mobile phase A, using acetonitrile as Mobile phase B, with Volume basis, according to the form below carry out gradient elution：The solvent is by 0.1% phosphate aqueous solution：Acetonitrile by volume 90:10 compositions.
- 2. analyzing detecting method as claimed in claim 1, it is characterised in that described sample solution concentration is 0.2mg/ml.
- 3. analyzing detecting method as claimed in claim 1, it is characterised in that described flow rate of mobile phase is 1.0ml/min, post Temperature is 30 DEG C.
- 4. analyzing detecting method as claimed in claim 1, it is characterised in that the mobile phase A is 0.1% phosphate aqueous solution.
- 5. analyzing detecting method as claimed in claim 1, it is characterised in that the eluent gradient elution is arranged to：
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112730702A (en) * | 2020-12-30 | 2021-04-30 | 南京百泽医药科技有限公司 | Method for determining related substances of 6-acetoxyl-7-methoxy-3H-quinazoline-4-one |
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| CN1764668A (en) * | 2002-12-24 | 2006-04-26 | 阿斯利康(瑞典)有限公司 | Phosphonooxy quinazoline derivatives and their pharmaceutical use |
| CN103864702A (en) * | 2014-02-27 | 2014-06-18 | 福建医科大学 | Method for preparing quinazolinone in water phase through microwave catalysis |
| CN105503872A (en) * | 2015-12-01 | 2016-04-20 | 北京普德康利医药科技发展有限公司 | Linagliptin impurity, and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112730702A (en) * | 2020-12-30 | 2021-04-30 | 南京百泽医药科技有限公司 | Method for determining related substances of 6-acetoxyl-7-methoxy-3H-quinazoline-4-one |
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