CN102375044A - Method for analyzing related substance from hydrochloric acid bendamustine intermediate Z6 - Google Patents
Method for analyzing related substance from hydrochloric acid bendamustine intermediate Z6 Download PDFInfo
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- CN102375044A CN102375044A CN2010102568603A CN201010256860A CN102375044A CN 102375044 A CN102375044 A CN 102375044A CN 2010102568603 A CN2010102568603 A CN 2010102568603A CN 201010256860 A CN201010256860 A CN 201010256860A CN 102375044 A CN102375044 A CN 102375044A
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Abstract
The invention relates to a method for analyzing a related substance from a hydrochloric acid bendamustine intermediate Z6 by using a reversed-phase high performance liquid chromatography, which is characterized in that a sample is dissolved by a mixed solvent of an organic solvent and acid water, the organic solvent and an ammonium salt aqueous solution are taken as a mobile phase, a gradient elution is carried out on an octadecylsilane chemically bonded silica chromatographic column. The method for analyzing the related substance is capable of easily and effectively solving the problem of separating the related substance from the hydrochloric acid bendamustine intermediate Z6.
Description
Technical field
The present invention relates to a kind of bulk drug bendamustine hydrochloride intermedium Z
6The analytical approach of related substance is particularly separated the method with quantitative measurement with high performance liquid chromatography to this intermedium related substance.
Background technology
" related substance (related substances) " alleged in Pharmaceutical Analysis is meant in the specific medication it is not main constituent, but the material relevant with composition.Since a kind of medicine from the synthesis material medicine to the relevant preparation of preparation; Again through storage, transportation, use; Experience one section comparatively complicated and very long process; During this period, each process all might produce relevant material, in producing, possibly bring initiation material, reagent, intermedium, accessory substance and isomeride etc. into; In storage and transportation, possibly produce special impurities such as catabolite, polymkeric substance or crystal transfer.For guaranteeing the safe and effective of medicine; Simultaneously also to consider the production actual conditions; Therefore, both at home and abroad to the research of medicine, can allow to contain the harmless or hypotoxic related substance of a threshold quantity; But bigger to toxicity, that can be detrimental to health, the invalid related substance that maybe can influence medicine stability then must strict control.Therefore the related substance detection is the important indicator of control drug quality.
When the synthesizing antineoplastic medicament bendamustine hydrochloride, its intermedium 4-(5-(two-hydroxyethyl) amino)-1-methyl isophthalic acid H-benzimidazolyl) uncle's ethyl butyrate is (hereinafter to be referred as Z
6) synthetic be important intermediate steps, Z
6The quality of quality is the key factor of medicine bendamustine hydrochloride quality assurance.And to Z
6The control of middle related substance is the important indicator that guarantees its quality.
At present, American Pharmacopeia USP32 version, European Pharmacopoeia EP6 version, British Pharmacopoeia, Japanese Pharmacopoeia and Chinese Pharmacopoeia are not all included bendamustine hydrochloride and intermedium Z thereof
6Method for determination related substances.
Summary of the invention
The purpose of this invention is to provide a kind of bendamustine hydrochloride intermedium Z
6(being 4-(5-(two-hydroxyethyl) amino)-1-methyl isophthalic acid H-benzimidazolyl) uncle's ethyl butyrate) related substance HPLC analytical method.Explore Z
6The various top conditions of related substance efficient liquid phase chromatographic analysis reach purpose easy, reliable, that effectively analyze.Through to intermedium Z
6Quality control, thereby reach the purpose that improves medicine bendamustine hydrochloride quality indirectly.
Bendamustine hydrochloride intermedium Z
6And five kinds of related substances see the following form:
Z
6Middle related substance
The inventor has found out suitable analysis condition through to chromatographic column, the equal condition repeated screening of flowing in the efficient liquid phase chromatographic analysis.For taking into account the needs that plurality of impurities is separated, selected the suitable buffer solution system and the crucial testing conditions such as ratio of moving phase, final method of establishing has reached the foregoing invention purpose.
Technical scheme of the present invention is following:
A kind of bendamustine hydrochloride intermedium Z
6The analytical approach of related substance; Adopt reversed-phased high performace liquid chromatographic; It is characterized in that mixed solvent sample dissolution with organic solvent and aqueous acid; The WS with organic solvent and ammonium salt or organic solvent and triethylamine-acetic acid is moving phase, on the octadecylsilane chemically bonded silica chromatographic column, carries out gradient elution; Said organic solvent is methyl alcohol or acetonitrile.
In the used mixed solvent of sample dissolution, the volume ratio of organic solvent and aqueous acid is 15: 85; Said acid is selected from one or more in phosphoric acid, acetic acid, the formic acid, and the consumption of acid is the pH to 3 that regulates mixed solvent.
Said ammonium salt aqueous solution is with acid for adjusting pH to 4.0～6.0; Said acid is selected from one or more in phosphoric acid, acetic acid, the formic acid; Said ammonium salt is selected from one or more in diammonium hydrogen phosphate, ammonium dihydrogen phosphate (ADP), the ammonium acetate.
Testing conditions is to detect wavelength: 233nm; Moving phase elution rate: 0.9～1.1ml/min; Chromatogram column temperature: 23～27 ℃; Sample feeding amount: 20 μ l.
The gradient of gradient elution is preferably in the said method:
| Time (min) | Acetonitrile (%) | Buffered saline solution (%) |
| 0 | 15 | 85 |
| 20 | 25 | 75 |
| 30 | 30 | 70 |
| 50 | 30 | 70 |
| 50.01 | 15 | 85 |
| 60 | 15 | 85 |
The inventor has found bendamustine hydrochloride intermedium Z through the screening to buffer salt in the HPLC analytical method
6And the tangible buffer solution system of impurity separating effect, and on the pH value is regulated, fully take into account the needs that several impurity effectively separate, and finally set up the HPLC gradient elution method, analytical cycle was controlled at 60 minutes.Consider the stability of sample solution simultaneously, different thinning agents are investigated, find suitable diluent, more perfect bendamustine hydrochloride intermedium Z
6The related substance analytical approach.
The effect and the advantage of this method are:
1, can effectively isolate bendamustine intermedium Z
6Related substance, impurity peaks separates with main peak fully, and method is effectively, reliably;
2, can be to Z
6Related substance carries out quantitatively, quality that can strict control final products bendamustine hydrochloride;
3, analysis time period is short, in 60 minutes, and can be with multiple known impurities and main peak baseline separation;
4, simple to operate.
Description of drawings:
Institute's drawings attached is working sample Z
6The high-efficient liquid phase chromatogram of middle related substance, wherein condition determination is respectively as follows:
Fig. 1: buffer solution is Potassium Hexafluorophosphate, the same the inventive method of other conditions;
Fig. 2: buffer solution is sodium dihydrogen phosphate, the same the inventive method of other conditions;
Fig. 3: buffer solution is potassium dihydrogen phosphate, the same the inventive method of other conditions;
Fig. 4: measure with the inventive method, use diammonium hydrogen phosphate to make buffer solution;
Fig. 5: measure with the inventive method, use ammonium acetate to make buffer solution;
Fig. 6: measure with the inventive method, regulate pH value of buffer solution=4.0;
Fig. 7: measure with the inventive method, regulate pH value of buffer solution=6.0;
Fig. 8: 0 hour mensuration need testing solution 3 related substance;
Fig. 9: the high-efficient liquid phase chromatogram (except that thinning agent was acetonitrile, all the other analytical approachs and the inventive method were together) of measuring need testing solution 1 related substance gained in 2 hours;
Figure 10: the high-efficient liquid phase chromatogram (divided by moving phase is outside the thinning agent, and all the other analytical approachs and the inventive method are together) of measuring need testing solution 2 related substance gained in 2 hours;
Figure 11: 2 hours mensuration need testing solution 3 related substances.
Embodiment:
Further explain the present invention with embodiment below, but protection scope of the present invention is not limited to employed analytical approach among these embodiment, adopts the method among the embodiment also can analyze multiple substituent similar compound.
Used Z among the embodiment
6And impurity standard items and bendamustine hydrochloride intermedium source is Huabang Pharmaceutical Co., Ltd., Chongqing.
The experimental apparatus that uses is Tianjin, island high performance liquid chromatograph (UV detecting device);
Except that indicating, the chromatographic column of using is VP-ODS 4.6 * 250mm, and 5 μ m, column temperature are 25 ℃; Sample size is 20 μ l; Flow velocity is 1.0ml/min, and sample concentration is 0.2mg/ml.The eluent gradient condition is following:
| Time (min) | Acetonitrile (%) | Buffered saline solution (%) |
| 0 | 15 | 85 |
| 20 | 25 | 75 |
| 30 | 30 | 70 |
| 50 | 30 | 70 |
| 50.01 | 15 | 85 |
| 60 | 15 | 85 |
Reagent and reagent: acetonitrile (chromatographically pure); Distilled water; Phosphoric acid (analyzing pure); Potassium Hexafluorophosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium acetate (it is pure to be analysis).
Following examples 1～4th are carried out Z with method of the present invention
6The example of analyzing; Experimental example 1～4th, the inventor carries out Z with additive method
6Analysis, as with the contrast of the inventive method.
Analysis condition in embodiment 1～2 and the experimental example 1～3, except that the buffered saline solution difference, all the other are identical.
Analysis condition among embodiment 1～2 and the embodiment 3～4, except that buffered saline solution pH value difference, all the other are identical.
Thinning agent: acetonitrile: water (phosphoric acid is transferred pH=3.0)=15: 85 (V/V);
Moving phase: acetonitrile: buffered saline solution (phosphoric acid is transferred pH)=15: 85 (V/V);
The contrast experiment
Need testing solution preparation: it is an amount of to take by weighing finished product, accurately claims surely, puts in the measuring bottle, adds thinning agent to suitable solubility, shakes up, and filters, and promptly gets.Sample introduction also writes down chromatogram.
Experimental result: buffer solution is that the experimental result of Potassium Hexafluorophosphate is seen accompanying drawing 1; Buffer solution is that the experimental result of sodium dihydrogen phosphate is seen accompanying drawing 2; Buffer solution is that the experimental result of potassium dihydrogen phosphate is seen accompanying drawing 3; Buffer solution is that the experimental result of diammonium hydrogen phosphate is seen accompanying drawing 4; Buffer solution is that the experimental result of ammonium acetate is seen accompanying drawing 5; The experimental result of ammonium dibasic phosphate aqueous solution adjustment pH=4.0 is seen accompanying drawing 6; The experimental result of ammonium dibasic phosphate aqueous solution adjustment pH=6.0 is seen accompanying drawing 7;
1) liquid chromatogram through different buffer solution relatively can know that main peak and each impurity separating effect are obvious when buffer solution is ammonium salt.
2) visible between pH value of buffer solution is adjusted at 4.0～6.0 the time by chromatogram, main peak can both reach effective the separation with each impurity.
Experimental example 4: different thinning agents are to bendamustine hydrochloride intermedium Z
6Determination of related substances
Moving phase: acetonitrile: ammonium dibasic phosphate aqueous solution (phosphoric acid is transferred pH=4.8)=15: 85 (V/V);
| Need testing | Thinning agent | |
| 1 | Acetonitrile | |
| 2 | Moving phase | |
| 3 ( |
Acetonitrile: water (pH=3.0 is transferred in acid)=15: 85 (v/v) |
Need testing solution preparation: it is an amount of to take by weighing finished product, accurately claims surely, puts in the measuring bottle, gets different diluting solvents respectively and is diluted to suitable solubility, shakes up, and filters, and promptly gets.Sample introduction also writes down chromatogram.The need testing solution of being prepared was placed after 2 hours, sample introduction (investigating stability and the main peak peak type of sample in different thinning agents).
Experimental result: the experimental result of 0 hour need testing solution 3 sample introduction is seen accompanying drawing 8; Need testing solution 1 (acetonitrile) is placed the experimental result of 2 hours sample introductions and is seen accompanying drawing 9; Need testing solution 2 (moving phase) is placed the experimental result of 2 hours sample introductions and is seen accompanying drawing 10, and the experimental result that need testing solution 3 is placed 2 hours sample introductions is seen accompanying drawing 11.
Visible by figure, be thinning agent with the acetonitrile, test sample main peak peak type is bad so abandon; 0 hour, Z
6aContent is 0.01%, places after 2 hours, with Z in the need testing solution of moving phase preparation
6aContent is 0.07%, and with acetonitrile: Z in the water need testing solution that (pH=3.0 is transferred in acid)=15: 85 (v/v) prepared
6aContent is 0.03%, so sample is at acetonitrile: more stable in the water (pH=3.0 is transferred in acid)=15: 85 (v/v), so thinning agent of selecting it to prepare as need testing solution.
Experimental example 5: bendamustine hydrochloride intermedium Z
6In the quantitative limit of each impurity
Chromatographic column: VP-ODS 4.6 * 250mm, 5 μ m
Flow velocity: 1.0ml/min column temperature: 25 ℃
Diluting solvent: acetonitrile: water (phosphoric acid is transferred pH=3.0)=15: 85 (V/V)
Detect wavelength: 233nm
Sample concentration: 0.2mg/ml
Moving phase: acetonitrile: ammonium dibasic phosphate aqueous solution (phosphoric acid is transferred pH=4.8)=15: 85 (V/V)
The eluent gradient condition is following:
| Time (min) | Acetonitrile % | Ammonium dibasic phosphate aqueous solution (phosphoric acid is transferred pH=4.8) % |
| 0 | 15 | ?85 |
| 20 | 25 | ?75 |
| 30 | 30 | ?70 |
| 50 | 30 | ?70 |
| 50.01 | 15 | ?85 |
| 60 | 15 | ?85 |
Each impurity quantitative limit solution preparation: take by weighing each impurity reference substance respectively, the accurate title, decide, and adds thinning agent to suitable concn, shakes up, and promptly gets, and sample introduction also writes down chromatogram.
The quantitative limit result of each impurity sees the following form in the bulk drug bendamustine hydrochloride:
Through the contrast of embodiment and experimental example, be moving phase with the WS of organic solvent and ammonium salt, bendamustine hydrochloride intermedium Z
6With intermedium Z
6In each impurity separating effect obvious; In the time of between pH value of buffer solution is adjusted at 4.0～6.0, main peak can both reach effective the separation with each impurity, satisfies and measures requirement.In addition, be that the dilution of sample solvent can influence chromatographic peak profile with pure acetonitrile, and sample solution is unstable; And select for use organic solvent and sour water to mix in 15: 85 by volume, and to regulate pH to 3 with acid (one or more in phosphoric acid, acetic acid, the formic acid) to make thinning agent, collection of illustrative plates peak shape symmetry and sample solution are stable.
Claims (5)
1. bendamustine hydrochloride intermedium Z
6The analytical approach of related substance; Adopt reversed-phased high performace liquid chromatographic; It is characterized in that the mixed solvent sample dissolution processed with organic solvent and aqueous acid; The WS with organic solvent and ammonium salt is moving phase, or is moving phase with the WS of organic solvent and triethylamine-acetic acid, on the octadecylsilane chemically bonded silica chromatographic column, carries out gradient elution; Said organic solvent is methyl alcohol or acetonitrile.
2. the described analytical approach of claim 1, in the used mixed solvent of sample dissolution, the volume ratio of organic solvent and aqueous acid is 15: 85; Said acid is selected from one or more in phosphoric acid, acetic acid, the formic acid, and the consumption of acid is the pH to 3 that regulates mixed solvent.
3. the described analytical approach of claim 1, said ammonium salt aqueous solution is with acid for adjusting pH to 4.0～6.0; Said acid is selected from one or more in phosphoric acid, acetic acid, the formic acid.
4. claim 1 or 3 described analytical approachs, said ammonium salt is selected from one or more in diammonium hydrogen phosphate, ammonium dihydrogen phosphate (ADP), the ammonium acetate.
5. the described analytical approach of claim 1, testing conditions are to detect wavelength: 233nm; Moving phase elution rate: 0.9～1.1ml/min; Chromatogram column temperature: 23～27 ℃; Sample feeding amount: 20 μ l.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103995063A (en) * | 2014-05-29 | 2014-08-20 | 四川汇宇制药有限公司 | Detection method for related substances in bendamustine hydrochloride |
| CN108445107A (en) * | 2018-04-11 | 2018-08-24 | 健进制药有限公司 | A kind of diluent and its detection method of the bendamustine hydrochloride in relation to substance |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101119708A (en) * | 2005-01-14 | 2008-02-06 | 赛福伦公司 | Bendamustine pharmaceutical compositions |
| WO2010063493A1 (en) * | 2008-12-03 | 2010-06-10 | Astellas Deutschland Gmbh | Oral dosage forms of bendamustine |
| WO2010063476A2 (en) * | 2008-12-03 | 2010-06-10 | Astellas Deutschland Gmbh | Solid dosage forms of bendamustine |
-
2010
- 2010-08-18 CN CN201010256860.3A patent/CN102375044B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101119708A (en) * | 2005-01-14 | 2008-02-06 | 赛福伦公司 | Bendamustine pharmaceutical compositions |
| WO2010063493A1 (en) * | 2008-12-03 | 2010-06-10 | Astellas Deutschland Gmbh | Oral dosage forms of bendamustine |
| WO2010063476A2 (en) * | 2008-12-03 | 2010-06-10 | Astellas Deutschland Gmbh | Solid dosage forms of bendamustine |
Non-Patent Citations (2)
| Title |
|---|
| IVANKA PENCHEVA ET AL: "HPLC study on the stability of bendamustine hydrochloride immobilized onto polyphosphoesters", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| 汤浩 等: "HPLC法测定盐酸苯达莫司汀含量及有关物质", 《药学与临床研究》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103995063A (en) * | 2014-05-29 | 2014-08-20 | 四川汇宇制药有限公司 | Detection method for related substances in bendamustine hydrochloride |
| CN108445107A (en) * | 2018-04-11 | 2018-08-24 | 健进制药有限公司 | A kind of diluent and its detection method of the bendamustine hydrochloride in relation to substance |
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|---|---|
| CN102375044B (en) | 2015-04-08 |
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