CN102375033A - High performance liquid chromatographic analysis method of bendamustine hydrochloride and its related substances - Google Patents
High performance liquid chromatographic analysis method of bendamustine hydrochloride and its related substances Download PDFInfo
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- CN102375033A CN102375033A CN2010102568730A CN201010256873A CN102375033A CN 102375033 A CN102375033 A CN 102375033A CN 2010102568730 A CN2010102568730 A CN 2010102568730A CN 201010256873 A CN201010256873 A CN 201010256873A CN 102375033 A CN102375033 A CN 102375033A
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Abstract
The invention relates to a method for determining related substances of bendamustine hydrochloride through high performance liquid chromatography. In the method, a sample to be detected is prepared into a sample solution with a mobile phase, and then reversed isocratic elution is carried out on an octadecylsilane bonded silica gel chromatographic column, with an organic solvent and a buffer solution as the mobile phase. The method of the invention effectively solves the problems in separation of related substances from bendamustine hydrochloride bulk drugs and detection thereof. The method provided in the invention has the advantages of simplicity, feasibility, and short analysis time cycle.
Description
Technical field
The present invention relates to the detection method of antineoplastic bendamustine hydrochloride and related substance thereof, the method for particularly analyzing with high performance liquid chromatography.
Background technology
In Pharmaceutical Analysis, it is the important indicator of control drug quality that related substance (related substances) detects.
Alleged " related substance " is meant in the specific medication it is not main constituent in the Pharmaceutical Analysis, but the material relevant with composition.A kind of medicine to the relevant preparation of preparation, again through storage, transportation, use, experience one section comparatively complicated and very long process from the synthesis material medicine.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 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.
Bendamustine hydrochloride (two (2-chloroethyl) amino of 1-methyl-5--2-benzimidazole butyrate hydrochlorate) is a kind of nitrogen mustards antineoplastic.The just listing in Europe in 2007 of this medicine is not recorded by European Pharmacopoeia and American Pharmacopeia as yet.For its related substance and Determination on content method, not only do not have concrete standard, and domestic and foreign literature do not see that play-by-play is arranged yet, there is not relevant information in various countries' patent database yet.
The related substance that mainly contains common in the bendamustine hydrochloride material medicine has eight kinds (seeing table 1), for guaranteeing the quality of medicine, need separate and measure its content.
Therefore, study and set up the analytical approach of bendamustine hydrochloride related substance, very necessary.
The structural formula of table 1 bendamustine hydrochloride and related substance thereof and chemical name
Summary of the invention
The purpose of this invention is to provide a kind of bendamustine hydrochloride related substance HPLC analytical method.Through exploring the various conditions of bendamustine hydrochloride related substance efficient liquid phase chromatographic analysis, reach purpose quick, easy, reliable, that effectively analyze.
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 8 kinds of needs that impurity separates in the finished product, 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:
The HPLC analytical method of related substance in a kind of bendamustine hydrochloride; Testing sample is mixed with the sample solution of 0.2mg/ml～1.5mg/ml with moving phase; On the octadecylsilane chemically bonded silica chromatographic column, carry out the anti-phase isocratic elution, moving phase is organic solvent and buffering solution; Said organic solvent is methyl alcohol or acetonitrile, and said buffer solution is the brine solution with acid for adjusting pH to 2.0～4.0; Said salt is selected from hexafluorophosphate, lauryl sulfate, phosphate, acetate or formates, and concentration is 0.01～0.05mol/L.
Said acid is selected from phosphoric acid, acetic acid, formic acid or trifluoroacetic acid.Preferred hexafluorophosphate potassium of said salt or lauryl sodium sulfate;
The volume ratio of organic solvent in moving phase is preferably 40%～45%.
Can adopt following testing conditions:
Sample detection wavelength: 220～245nm; Moving phase elution rate: 0.8～1.2ml/min; Chromatogram column temperature: 15～35 ℃; Sample size: 20 μ l.
The effect and the advantage of this method are:
1, can effectively isolate the impurity in the bendamustine hydrochloride bulk drug, method effectively, reliably;
2, can carry out quantitatively the impurity in the bendamustine hydrochloride bulk drug, can strictly control the bendamustine hydrochloride quality;
3, analysis time period is short, in 40 minutes, and can be with eight known impurities and main peak baseline separation;
4, simple to operate.
Description of drawings:
Fig. 1: the inventive method system specificity experiment chromatogram (each peak marks by retention time among the figure, and main peak is a bendamustine hydrochloride, and what wherein indicate especially with literal is each known impurities peak that possibly occur in the bendamustine hydrochloride);
Fig. 2～Figure 10, Figure 12 measure the resulting high-efficient liquid phase chromatogram of bendamustine hydrochloride related substance with the inventive method, are labeled as among the figure to detect impurity E and the unknown impuritie 1～4 that obtains, and detect basic condition and are: 25 ℃ of column temperatures; Sample introduction concentration 1.0mg/ml; Flow velocity: 1.0ml/min; Detect wavelength 233nm;
Fig. 3～Figure 10 adjusts the collection of illustrative plates that records behind the relevant parameter on this basis, wherein:
Fig. 3: flow rate of mobile phase is 0.8ml/min;
Fig. 4: flow rate of mobile phase is 1.2ml/min;
Fig. 5: the chromatographic column column temperature is 15 ℃;
Fig. 6: the chromatographic column column temperature is 35 ℃;
Fig. 7: moving phase ratio A: B (V: V)=45: 55;
Fig. 8: moving phase ratio A: B (V: V)=40: 60; Fig. 9: pH value of buffer solution is 2.0;
Figure 10: pH value of buffer solution is 4.0;
Figure 11: each impurity ultraviolet spectrogram of bendamustine hydrochloride;
Figure 12: different sample concentration bendamustine hydrochlorides, wherein the sample introduction concentration of A, B, C collection of illustrative plates be respectively 0.8,1.0,1.5mg/ml.
Embodiment:
Following examples 1～9th are carried out the example that bendamustine hydrochloride is analyzed with method of the present invention;
Experimental example 1～2nd, the inventor has carried out the analysis of bendamustine hydrochloride with other two kinds of methods, as with the contrast of the inventive method.
The filler of the chromatography column of the composition of the moving phase of employed chromatographic resolution, moving phase and use and chromatography column etc. among the embodiment is only as further explaining the present invention.But protection scope of the present invention is not limited to these.
Main experimental apparatus and chromatographic condition:
The experimental apparatus that uses in following examples is Tianjin, island LC2010A type high performance liquid chromatograph; Automatic sampler; The UV detecting device, the control of chromatographic fractionation system and record are accomplished (except screening of related substance wavelength and the concentration screening test: use instrument to be Agilent 1200 type high performance liquid chromatographs, automatic sampler by Tianjin, island LC Solution chromatogram management system; The DAD detecting device, the control of chromatographic fractionation system and record are accomplished by Agilent Chemstation chromatogram management system).
Except that indicating, the chromatographic column of using is VP-ODS C
184.6 * 250mm 5 μ m; Column temperature is 25 ℃; Sample size is 20 μ l; Flow velocity: 1.0ml/min; Detect wavelength 233nm.
Reagent: acetonitrile, chromatographically pure; Pure water is analyzed pure; Potassium Hexafluorophosphate, Aladdin reagent
Methyl alcohol, chromatographically pure; Trifluoroacetic acid is analyzed pure
Sample sees the following form:
The situation of bendamustine hydrochloride and related substance thereof
Embodiment 1: bendamustine hydrochloride determination of related substances ()
1) bendamustine hydrochloride determination of related substances-system's specificity experiment
Chromatographic condition: moving phase: A: acetonitrile; B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH to 3.0); A: B=425: 575 (V: V)
The sample solution preparation: (A～H) an amount of with finished product, the accurate title, decide, and puts in the same measuring bottle, adds thinning agent to suitable concn, shakes up, and filters, and promptly gets to take by weighing each impurity standard items respectively.Sample introduction also writes down chromatogram.
Experimental result: see accompanying drawing 1, the main peak retention time is 12.3min, main peak and each impurity peaks good separation, and each impurity peaks all can baseline separation.
2) quantitative limit of each impurity and detectability detect in the bulk drug bendamustine hydrochloride:
Chromatographic condition: moving phase: A: acetonitrile B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH 3.0) A: B=425: 575 (V: V)
Sample solution preparation: it is an amount of to take by weighing each impurity reference substance respectively, accurately claims surely, adds moving phase to suitable concn, shakes up, and filters, and promptly gets.Sample introduction also writes down chromatogram.
The quantitative limit result of each impurity sees the following form in the bulk drug bendamustine hydrochloride:
The detectability result of each impurity sees the following form in the bulk drug bendamustine hydrochloride:
3) bendamustine hydrochloride determination of related substances-sample detection
Moving phase: A: acetonitrile; B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH to 3.0); A: B=425: 575 (V: V)
Sample solution preparation: it is an amount of to take by weighing finished product, accurately claims surely, puts in the same measuring bottle, adds moving phase to suitable concn, shakes up, and promptly gets (1.0mg/ml).Sample introduction also writes down chromatogram.
Experimental result: see accompanying drawing 2, the main peak retention time is 12.3min, each impurity peaks and main peak good separation, and each impurity peaks (unknown impuritie 1-4) all can baseline separation.
Moving phase: A: acetonitrile; B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH);
Sample solution preparation: it is an amount of to take by weighing finished product, accurately claims surely, puts in the same measuring bottle, adds moving phase to suitable concn, shakes up, and promptly gets.Sample introduction also writes down chromatogram.
Experimental result: accompanying drawing 3～10, each impurity peaks and main peak good separation, and each impurity peaks (unknown impuritie 1-4) all can baseline separation.
Instrument: Agilent 1200 type high performance liquid chromatographs (DAD detecting device)
Moving phase: A: acetonitrile; B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH);
Sample solution preparation: it is an amount of to take by weighing finished product, accurately claims surely, puts in the same measuring bottle, adds moving phase to suitable concn, shakes up, and promptly gets.Sample introduction also writes down chromatogram.
Experimental result:
1) by accompanying drawing 11, extract ultraviolet spectrum and show that each impurity is the homolog of finished product, uv-absorption maximum wavelength is identical.When the detection wavelength was respectively 220nm, 233nm and 245nm, there was horizontal basically identical in each impurity.
2) by accompanying drawing 12, sample concentration is respectively 0.8,1.0, during 1.5mg/ml; Each impurity detection level basically identical.
| Concentration | Impurity E | |
|
| 0.8mg/ml | 0.05 | 0.05 | 0.02 |
| 1.0mg/ml | 0.04 | 0.05 | 0.02 |
| 1.5mg/ml | 0.04 | 0.05 | 0.02 |
Claims (5)
1. the HPLC analytical method of related substance in the bendamustine hydrochloride; Testing sample is mixed with the sample solution of 0.2mg/ml～1.5mg/ml with moving phase; On the octadecylsilane chemically bonded silica chromatographic column, carry out the anti-phase isocratic elution, moving phase is organic solvent and buffering solution; Said organic solvent is methyl alcohol or acetonitrile, and said buffer solution is the brine solution with acid for adjusting pH to 2.0～4.0; Said salt is selected from hexafluorophosphate, lauryl sulfate, phosphate, acetate or formates, and concentration is 0.01mol/L～0.05mol/L.
2. analytical approach according to claim 1, the used acid of said adjusting pH is selected from phosphoric acid, acetic acid, formic acid or trifluoroacetic acid.
3. analytical approach according to claim 1, said salt are hexafluorophosphate potassium or lauryl sodium sulfate.
4. analytical approach according to claim 1, the volume ratio of said organic solvent in moving phase is 40%～45%.
5. analytical approach according to claim 1, testing conditions is: detect wavelength: 220～245nm; Moving phase elution rate: 0.8～1.2ml/min; Chromatogram column temperature: 15～35 ℃; Sample size: 20 μ l.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103351347A (en) * | 2013-07-29 | 2013-10-16 | 东南大学 | Preparation method of impurity DCE in bendamustine hydrochloride |
| 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 |
| CN105085405A (en) * | 2014-05-06 | 2015-11-25 | 江苏先声药业有限公司 | Preparation method and application of 4-[1-methyl-5-(2-chloroethyl-2-ethoxyl)amino-2-benzimidazolyl]butyric acid hydrochloride |
| CN107305199A (en) * | 2016-04-19 | 2017-10-31 | 重庆华邦制药有限公司 | Two components and the method about material in separation determination azelastine hydrochloride fluticasone propionate compound nasal spray |
| 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 |
| CN112394134A (en) * | 2019-08-14 | 2021-02-23 | 江苏晶立信医药科技有限公司 | Detection method of related substances of cimetidine raw material medicine |
| CN115356413A (en) * | 2022-08-17 | 2022-11-18 | 博诺康源(北京)药业科技有限公司 | Method for detecting scopolamine hydrobromide related substances |
-
2010
- 2010-08-18 CN CN201010256873.0A patent/CN102375033B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| IVANKA PENCHENVA等: "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》 * |
| 陈栋编译: "盐酸苯达莫司汀(bendamustine hydrochloride)", 《中国药物化学杂志》 * |
| 高丽梅等: "盐酸苯达莫司汀的合成", 《中国新药杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103351347A (en) * | 2013-07-29 | 2013-10-16 | 东南大学 | Preparation method of impurity DCE in bendamustine hydrochloride |
| CN105085405A (en) * | 2014-05-06 | 2015-11-25 | 江苏先声药业有限公司 | Preparation method and application of 4-[1-methyl-5-(2-chloroethyl-2-ethoxyl)amino-2-benzimidazolyl]butyric acid hydrochloride |
| 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 |
| CN107305199A (en) * | 2016-04-19 | 2017-10-31 | 重庆华邦制药有限公司 | Two components and the method about material in separation determination azelastine hydrochloride fluticasone propionate compound nasal spray |
| CN107305199B (en) * | 2016-04-19 | 2021-04-13 | 重庆华邦制药有限公司 | Method for separating and measuring two components and related substances in compound nasal spray of azelastine hydrochloride and fluticasone propionate |
| 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 |
| CN112394134A (en) * | 2019-08-14 | 2021-02-23 | 江苏晶立信医药科技有限公司 | Detection method of related substances of cimetidine raw material medicine |
| CN115356413A (en) * | 2022-08-17 | 2022-11-18 | 博诺康源(北京)药业科技有限公司 | Method for detecting scopolamine hydrobromide related substances |
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