CN105424842A - Method for detecting Afatinib and relevant substances thereof - Google Patents
Method for detecting Afatinib and relevant substances thereof Download PDFInfo
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- CN105424842A CN105424842A CN201511004462.1A CN201511004462A CN105424842A CN 105424842 A CN105424842 A CN 105424842A CN 201511004462 A CN201511004462 A CN 201511004462A CN 105424842 A CN105424842 A CN 105424842A
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- Prior art keywords
- buddhist nun
- detection method
- mobile phase
- acetonitrile
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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 invention relates to a method for detecting Afatinib, relevant substances thereof, and especially cis-isomers thereof. The method comprises the following steps: carrying out gradient elution by using a high performance liquid chromatograph, wherein Phenomenex C18 (250mm*4.6mm, 5[mu]m) is adopted as a chromatographic column, an aqueous solution containing 0.2% of formic acid and 0.1% of trifluoroacetic acid is adopted as a mobile phase A, acetonitrile is adopted as a mobile phase B, the detection wavelength is 255-265nm, and the column temperature is 35-45DEG C; and carrying out gradient elution under certain conditions. The method can effectively separate and detect Afatinib, relevant substances thereof, and especially cis-isomers thereof.
Description
Technical field
The Ah method of the present invention relates to replaces the detection method of Buddhist nun and related substance thereof, and especially Ah method belongs to pharmaceutical technology sectors for the detection method of Buddhist nun's cis-isomer.
Background technology
Ah method is the irreversible inhibitor of a kind of EGF-R ELISA (EGFR) and human epidermal growth factor receptor 2 (Her-2) tyrosine kinase for Buddhist nun (Afatinib), and its 2-maleate can be used for the diseases such as treatment advanced Non-small cell lung (NSCLC).Chemistry (2E)-4-by name [(the chloro-4-fluorophenyl of 3-) the is amino]-6-{ [4-(N of this compound, N-dimethylamino)-1-oxo-2-butene-1-Ji] amino-7-((3S)-tetrahydrofuran-3-base oxygen base)-quinazoline, structural formula is as follows:
WO2007/085638A discloses the preparation method of a kind of Ah method for Buddhist nun, and its synthetic route is as follows:
Gained Ah method reacts with maleic acid after can refining further for Buddhist nun's crude product, and the Ah method that generates is for Buddhist nun's 2-maleate.
Ah method replaces Buddhist nun (API) containing double bond and chiral carbon structure and less stable, therefore Ah method easily produces impurity for Buddhist nun and 2-maleate thereof in preparation or storage process, comprise (S, Z)-N-{4-[(the chloro-4-fluorophenyl of 3-) is amino]-7-[(tetrahydrofuran-3-base) oxygen base] quinazoline-6-base }-4-(dimethylamino) but-2-enamides (RC1), (R, E)-N-{4-[(the chloro-4-fluorophenyl of 3-) is amino]-7-[(tetrahydrofuran-3-base) oxygen base] quinazoline-6-base }-4-(dimethylamino) but-2-enamides (RC2), (S, Z)-N-{4-[(the chloro-4-fluorophenyl of 3-) is amino]-7-[(tetrahydrofuran-3-base) oxygen base] quinazoline-6-base }-4-(hydroxyl) but-2-enamides (RC3), (S)-N4-(the chloro-4-fluorophenyl of 3-)-7-[(tetrahydrofuran-3-base) oxygen base] quinazoline-4, 6-diamines (RC4), dimethylamine (RC5) etc., wherein, cis-isomer impurity RC1 and close with target product character and be difficult to be separated, detect.Process contaminants (S)-N-{4-[(the chloro-4-fluorophenyl of 3-) is amino]-7-[(tetrahydrofuran-3-base) oxygen base] quinazoline-6-base is also related in the synthetic method of WO2007/085638A }-4-carbamyl-phosphonic acids diethyl ester (RC6).
WO2013/052157A discloses the method for detecting purity of a kind of Ah method for Buddhist nun's 2-maleate, it adopts superelevation liquid phase chromatography to realize, and chromatographic column is PhenomenexC18100A (150mm × 4.6mm, 2.6 μm), determined wavelength is 260/4nm, Ref550/100nm, column temperature is 40 DEG C, and flow velocity is 0.7mL/min, sample size is 1 μ L, mobile phase is acetonitrile is mobile phase A, and 0.2% formic acid+0.1% hyptafluorobutyric acid pH2 is Mobile phase B, and its condition of gradient elution is as follows:
The method instrument apparatus expensive and not there is general adaptability, and require harsh to agents useful for same, chromatographic column, wherein hyptafluorobutyric acid is expensive.In addition, it is few for the dopant species that can detect during Buddhist nun that the method detects Ah method, and degree of separation is poor.As can be seen here, the practicality of the method and poor for applicability.
Therefore, one is needed to be suitable for separation and detection Ah method for Buddhist nun and related substance thereof, the especially method of its cis-isomer, effectively to control the quality of medicine.
Summary of the invention
The invention provides the detection method that a kind of Ah method replaces Buddhist nun and related substance thereof, carry out gradient elution on high performance liquid chromatograph, chromatographic column is octadecyl silane chromatographic column, mobile phase be containing 0.2% formic acid, 0.1% trifluoroacetic acid aqueous solution be mobile phase A, acetonitrile is Mobile phase B, and determined wavelength is 255nm ~ 265nm, column temperature is 35 ~ 45 DEG C, wherein, condition of gradient elution is as follows
In certain preferred embodiments of the present invention, described related substance is the cis-isomer of Ah method for Buddhist nun.
In certain preferred embodiments of the present invention, described octadecyl silane chromatographic column is PhenomenexC18 (250mm × 4.6mm, 5 μm).
Inventor finds that determined wavelength can select any wavelength within the scope of 255nm ~ 265nm, and all can meet detection Ah method being replaced to Buddhist nun and related substance thereof.In certain preferred embodiments of the present invention, described determined wavelength is 260nm.
In other preferred embodiments of the present invention, described column temperature is 40 DEG C.
In certain preferred embodiments of the present invention, described flow velocity is 1.0mL/min.
In the present invention, described " flow velocity " ± 0.1mL/min, " mobile phase ratio " ± 2%, " determined wavelength " ± 2nm on enforcement nothing of the present invention impact, and all fall within the scope of protection of the present invention.
Inventor also finds, thinning agent of the present invention can select any solubilized sample and to the glitch-free solvent of system, such as water, 30% acetonitrile, acetonitrile, methyl alcohol, or volume ratio is the aqueous solution containing 0.2% formic acid, 0.1% trifluoroacetic acid of 85:15: the mixed solution of acetonitrile, and all can meet detection Ah method being replaced to Buddhist nun and related substance thereof.
In the present invention, described " Ah method is for Buddhist nun ", when clearly not distinguishing, can be that Ah method replaces the sterling of Buddhist nun, bulk drug, composite, Ah method replaces Buddhist nun as the product of major component for the salt (if maleic acid Ah method is for Buddhist nun) etc. of Buddhist nun using Ah method.Assay method of the present invention is all applicable to the quality control of the said goods, and the assay method of the related substance of these products is also protected in protection scope of the present invention.
In the present invention, described " related substance " refers in medicine preparation and storage process, to the issuable impurity of synthetic method (comprising the organic impurities such as intermediate, accessory substance).Wherein, Ah method comprises the potential impurity relevant with its structure such as its cis-isomer (RC1) for the related substance of Buddhist nun, such as its degradation impurity RC3, RC4, RC5.
Detection method of the present invention, can Buddhist nun and cis-isomer and the potential impurity relevant with its structure thereof be replaced effectively to be separated Ah method, degree of separation be good, thus effectively can control the quality of Ah method for Buddhist nun.Method of the present invention can be separated the Ah method of detecting simply, fast and accurately for Buddhist nun and cis-isomer thereof and the potential impurity relevant with its structure, and specificity is strong, highly sensitive, good stability, favorable reproducibility, precision are high, peak type is good, may be used for the effective control in actual production.
Accompanying drawing explanation
Fig. 1 is the chromatogram of embodiment 1 blank solution.
Fig. 2 is the chromatogram of maleic acid Ah method for Buddhist nun's contrast solution of embodiment 1.
Fig. 3 is the chromatogram of the degree of separation solution of embodiment 1.
Fig. 4 is the chromatogram of maleic acid Ah method for Buddhist nun's sample of embodiment 1.
Fig. 5 is the chromatogram of maleic acid Ah method for Buddhist nun's reference substance of embodiment 2.
Fig. 6 is the chromatogram of maleic acid Ah method for Buddhist nun's sample of embodiment 2.
Fig. 7 is the chromatogram of maleic acid Ah method for Buddhist nun's reference substance of embodiment 3.
Fig. 8 is the chromatogram of maleic acid Ah method for Buddhist nun's sample of embodiment 3.
Fig. 9 is the chromatogram of maleic acid Ah method for Buddhist nun's sample of comparative example.
Specific embodiments
In order to make those skilled in the art understand technical scheme of the present invention better, below disclose further some non-limiting embodiments the present invention is described in further detail.
Reagent used in the present invention all can be buied from the market.
Embodiment 1
(1) testing conditions
Instrument: waters2695 high performance liquid chromatograph, 2996PDA detecting device, determined wavelength: 260nm;
Chromatographic column: PhenomenexC18 (250mm × 4.6mm, 5 μm) chromatographic column;
Thinning agent: 30% acetonitrile;
Mobile phase: the aqueous solution (trifluoroacetic acid solution of the formic acid and 1ml of getting 2ml mixes in 1000ml water) of 0.2% formic acid+0.1% trifluoroacetic acid is mobile phase A, and acetonitrile is Mobile phase B;
Flow velocity: 1.0ml/min;
Column temperature: 40 DEG C;
Sample size: 10uL;
Linear gradient elution is carried out by table 1.
Table 1
(2) detecting step
the preparation of degree of separation solution:accurately take cis-isomer (RC1), maleic acid Ah method mixes according to a certain amount of ratio, as degree of separation solution for Buddhist nun, RC3 and RC4.
the preparation of reference substance solution:get maleic acid Ah method appropriate for Buddhist nun's reference substance, add thinning agent dilution and make the solution of every 1ml containing 0.5 μ g, product solution in contrast.
the preparation of need testing solution:get maleic acid Ah method and be about 25mg for Buddhist nun's sample, accurately weighed, in 50mL measuring bottle, dissolve with thinning agent and be diluted to scale, shaking up, as need testing solution.
Precision measures each 10 μ L of blank solution, reference substance solution, degree of separation solution and need testing solution respectively, injection liquid chromatography, and record chromatogram, is shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4.Result shows: in fig. 2, and each peak is for Ah method is for Buddhist nun API
(t
r=17.421min); In figure 3, each peak is maleic acid (t
r=3.4min), cis-isomer RC1 (t
r=16.227min), Ah method is for Buddhist nun API (t
r=17.419min), RC3 (t
r=18.165min), RC4 (t
r=23.295min).
Embodiment 2
According to the detection method that embodiment 1 is similar, wherein, determined wavelength replaces with 255nm.The chromatogram of its reference substance is shown in Fig. 5, the chromatogram of test sample is shown in Fig. 6.
Embodiment 3
According to the detection method that embodiment 1 is similar, wherein, determined wavelength replaces with 265nm.The chromatogram of its reference substance is shown in Fig. 7, the chromatogram of test sample is shown in Fig. 8.
method validation test findings
According to " Chinese Pharmacopoeia " version governing principle 9101 drug standard analytical approach verification guide principle part in 2015, carry out Method validation test, under the chromatographic condition that wherein durability is investigated, comprise flow velocity ± 0.1mL/min, column temperature ± 5 DEG C, mobile phase ratio ± 2%, wavelength ± 2nm.The results are shown in Table 2.
table 2
Comparative example
With reference to the method for WO2013/052157A, sour water is replaced with mutually the solution of 0.2% more conventional formic acid+0.1% trifluoroacetic acid, meanwhile, the method under UPLC is changed into HPLC.
(1) testing conditions
Instrument: waters2695 high performance liquid chromatograph, 2996PDA detecting device, determined wavelength: 260nm;
Chromatographic column: PhenomenexC18 (250mm × 4.6mm, 5 μm) chromatographic column;
Thinning agent: 30% acetonitrile;
Mobile phase: the aqueous acid (trifluoroacetic acid solution of the formic acid and 1ml of getting 2ml mixes in 1000ml water) of 0.2% formic acid+0.1% trifluoroacetic acid is mobile phase A, and acetonitrile is Mobile phase B;
Flow velocity: 1.0ml/min;
Column temperature: 40 DEG C;
Sample size: 10uL;
Linear gradient elution program is carried out by table 3.
Table 3
Precision measures the need testing solution 10 μ L of embodiment 1, injection liquid chromatography, and record chromatogram, is shown in Fig. 3.Result shows: in fig .9, and each peak is maleic acid (t
r=2.5min), cis-isomer RC1 (t
r=7.287min), Ah method is for Buddhist nun API (t
r=17.419min), RC3 (t
r=10.593min), and RC4 and two the mutual juxtaposition in unknown impuritie peak, degree of separation is 0.432min.
Claims (7)
1. an Ah method is for the detection method of Buddhist nun and related substance thereof, it is characterized in that, high performance liquid chromatograph carries out gradient elution, and chromatographic column is octadecyl silane chromatographic column, mobile phase be containing 0.2% formic acid, 0.1% trifluoroacetic acid aqueous solution be mobile phase A, acetonitrile is Mobile phase B, determined wavelength is 255nm ~ 265nm, and column temperature is 35-45 DEG C, wherein, condition of gradient elution is as follows
2. detection method according to claim 1, is characterized in that, described related substance is the cis-isomer of Ah method for Buddhist nun.
3. detection method according to claim 1, is characterized in that, described octadecyl silane chromatographic column is PhenomenexC18 (250mm × 4.6mm, 5 μm).
4. according to the detection method of claim 1 or 2, it is characterized in that, described determined wavelength is 260nm.
5. according to the detection method of claim 1 or 2, it is characterized in that, described column temperature is 40 DEG C.
6. according to the detection method of claim 1 or 2, it is characterized in that, described flow velocity is 1.0mL/min.
7. according to the detection method of claim 1 or 2, it is characterized in that, the thinning agent of described detection method is selected from water, 30% acetonitrile, acetonitrile, methyl alcohol, or volume ratio is the aqueous solution containing 0.2% formic acid, 0.1% trifluoroacetic acid of 85:15: the mixed solution of acetonitrile.
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| CN201511004462.1A CN105424842A (en) | 2015-12-29 | 2015-12-29 | Method for detecting Afatinib and relevant substances thereof |
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Cited By (6)
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| CN106290620A (en) * | 2016-07-31 | 2017-01-04 | 合肥远志医药科技开发有限公司 | A kind of detection method of Afatinib crude drug isomer |
| CN106568853A (en) * | 2016-12-30 | 2017-04-19 | 山东康美乐医药科技有限公司 | A method of detecting impurities in a lapatinib intermediate |
| CN107490631A (en) * | 2016-06-10 | 2017-12-19 | 山东新时代药业有限公司 | A kind of analyzing detecting method of afatinib intermediate |
| CN107490646A (en) * | 2016-06-10 | 2017-12-19 | 山东新时代药业有限公司 | A kind of method with high effective liquid chromatography for measuring afatinib intermediate content |
| CN108663442A (en) * | 2017-12-30 | 2018-10-16 | 南京海融制药有限公司 | A kind of inspection method of the Alfacalcidol tablet in relation to substance |
| CN113917043A (en) * | 2021-11-04 | 2022-01-11 | 南京安杰新生物医药有限公司 | Detection method of afatinib maleate genotoxic impurities |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107490631A (en) * | 2016-06-10 | 2017-12-19 | 山东新时代药业有限公司 | A kind of analyzing detecting method of afatinib intermediate |
| CN107490646A (en) * | 2016-06-10 | 2017-12-19 | 山东新时代药业有限公司 | A kind of method with high effective liquid chromatography for measuring afatinib intermediate content |
| CN106290620A (en) * | 2016-07-31 | 2017-01-04 | 合肥远志医药科技开发有限公司 | A kind of detection method of Afatinib crude drug isomer |
| CN106290620B (en) * | 2016-07-31 | 2019-02-15 | 南京臣功制药股份有限公司 | A kind of detection method of Afatinib bulk pharmaceutical chemicals isomers |
| CN106568853A (en) * | 2016-12-30 | 2017-04-19 | 山东康美乐医药科技有限公司 | A method of detecting impurities in a lapatinib intermediate |
| CN108663442A (en) * | 2017-12-30 | 2018-10-16 | 南京海融制药有限公司 | A kind of inspection method of the Alfacalcidol tablet in relation to substance |
| CN108663442B (en) * | 2017-12-30 | 2021-03-30 | 南京海融制药有限公司 | Method for checking related substances of alfacalcidol tablet |
| CN113917043A (en) * | 2021-11-04 | 2022-01-11 | 南京安杰新生物医药有限公司 | Detection method of afatinib maleate genotoxic impurities |
| CN113917043B (en) * | 2021-11-04 | 2023-10-13 | 南京安杰新生物医药有限公司 | Detection method of afatinib maleate genotoxic impurities |
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Application publication date: 20160323 |