CN102375033B - 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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- CN102375033B CN102375033B CN201010256873.0A CN201010256873A CN102375033B CN 102375033 B CN102375033 B CN 102375033B CN 201010256873 A CN201010256873 A CN 201010256873A CN 102375033 B CN102375033 B CN 102375033B
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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, particularly carry out the method analyzed by high performance liquid chromatography.
Background technology
In Pharmaceutical Analysis, it is the important indicator controlling drug quality that related substance (related substances) detects.
" related substance " alleged in Pharmaceutical Analysis refers in certain drug it is not main constituent, but the material relevant to composition.Medicine from synthesis material medicine to preparing a relevant preparation, then through storage, transport, use, experience one section of comparatively complexity and very long process.During this period, each process all likely produces relevant material, may bring initiation material, reagent, intermediate, accessory substance and isomeride etc. in producing; In storage and transportation, the special impurities such as catabolite, polymkeric substance or crystal transfer may be produced.For ensureing the safe and effective of medicine, also to consider production actual conditions simultaneously, therefore the harmless or hypotoxic related substance containing a threshold quantity can be allowed, but comparatively large to toxicity, and that can be detrimental to health, the invalid related substance that maybe can affect medicine stability then must strictly control.
Bendamustine hydrochloride (two (2-chloroethyl) amino-2-benzimidazole butyrate hydrochlorate of 1-methyl-5-) is a kind of nitrogen mustards antineoplastic.This medicine 2007 just in Europe listing, not yet by European Pharmacopoeia and American Pharmacopeia record.For the assay method of its related substance and content, not only there is no concrete standard, and domestic and foreign literature is also there are no play-by-play, also without relevant information in various countries' patent database.
The related substance that mainly contains common in bendamustine hydrochloride material medicine has eight kinds (see table 1), for ensureing the quality of medicine, needs carry out being separated 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 object of this invention is to provide a kind of bendamustine hydrochloride related substance HPLC analytical method.By exploring the various conditions of bendamustine hydrochloride related substance efficient liquid phase chromatographic analysis, reaching quick, easy, reliable, effectively carrying out the object analyzed.
Inventor, by chromatographic column, equal condition repeated screening of flowing in efficient liquid phase chromatographic analysis, has found out suitable analysis condition.For taking into account the needs of 8 kinds of magazins' layout in finished product, have selected the crucial testing conditions such as the ratio of suitable buffer solution system and mobile phase, final method of establishing reaches foregoing invention object.
Technical scheme of the present invention is as follows:
The HPLC analytical method of related substance in a kind of bendamustine hydrochloride, testing sample mobile phase is mixed with the sample solution of 0.2mg/ml ~ 1.5mg/ml, octadecylsilane chemically bonded silica chromatographic column carries out Reversed Phase Isocratic wash-out, and mobile phase is organic solvent and buffer solution; Described organic solvent is methyl alcohol or acetonitrile, and described buffer solution is the brine solution with acid for adjusting pH to 2.0 ~ 4.0; Described salt is selected from hexafluorophosphate, lauryl sulfate, phosphate, acetate or formates, and concentration is 0.01 ~ 0.05mol/L.
Described acid is selected from phosphoric acid, acetic acid, formic acid or trifluoroacetic acid.The preferred hexafluorophosphate potassium of described salt or lauryl sodium sulfate;
The volume ratio of organic solvent in mobile phase is preferably 40% ~ 45%.
Following testing conditions can be adopted:
Sample detection wavelength: 220 ~ 245nm; Mobile phase elution rate: 0.8 ~ 1.2ml/min; Chromatogram column temperature: 15 ~ 35 DEG C; Sample size: 20 μ l.
Effect and the advantage of this method are:
1, effectively can isolate the impurity in bendamustine hydrochloride bulk drug, method effectively, reliably;
2, can carry out quantitatively, can strictly controlling bendamustine hydrochloride quality to the impurity in bendamustine hydrochloride bulk drug;
3, analysis time period is short, in 40 minutes, and can by eight known impurities and main peak baseline separation;
4, simple to operate.
Accompanying drawing illustrates:
Fig. 1: the inventive method system specificity experiment chromatogram (in figure, each peak marks by retention time, and main peak is bendamustine hydrochloride, and what wherein indicate especially with word is each known impurities peak that may occur in bendamustine hydrochloride);
Fig. 2 ~ Figure 10, Figure 12 are the high-efficient liquid phase chromatograms obtained with the inventive method mensuration bendamustine hydrochloride related substance, are labeled as the impurity E and unknown impuritie 1 ~ 4 that detect and obtain in figure, and detecting basic condition is: column temperature 25 DEG C; Sample introduction concentration 1.0mg/ml; Flow velocity: 1.0ml/min; Determined wavelength 233nm;
Fig. 3 ~ Figure 10 is the collection of illustrative plates recorded after adjusting 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: chromatographic column column temperature is 15 DEG C;
Fig. 6: chromatographic column column temperature is 35 DEG C;
Fig. 7: mobile phase ratio A: B (V: V)=45: 55;
Fig. 8: mobile 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 hydrochloride, 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 ~ 9 are the examples of carrying out bendamustine hydrochloride analysis by method of the present invention;
Experimental example 1 ~ 2 is that inventor has carried out the analysis of bendamustine hydrochloride, as the contrast with the inventive method by other two kinds of methods.
The mobile phase of the chromatographic resolution used in embodiment, the composition of mobile phase and the chromatography column of use and the filler etc. of chromatography column, only as explaining the present invention further.But protection scope of the present invention, is not limited to these.
Major experimental instrument and chromatographic condition:
The experimental apparatus used in following examples is Shimadzu LC2010A type high performance liquid chromatograph, automatic sampler, UV detecting device, the control of chromatographic fractionation system and record are completed by Shimadzu LC Solution Chromatography Management System that (related substance wavelength screens and except concentration screening test: use instrument is Agilent 1200 type high performance liquid chromatograph, automatic sampler, DAD detecting device, control and the record of chromatographic fractionation system are completed by Agilent Chemstation Chromatography Management System).
Except indicating, the chromatographic column of use is VP-ODS C
184.6 × 250mm 5 μm; Column temperature is 25 DEG C; Sample size is 20 μ l; Flow velocity: 1.0ml/min; Determined wavelength 233nm.
Reagent: acetonitrile, chromatographically pure; Pure water, analyzes pure; Potassium Hexafluorophosphate, Aladdin reagent
Methyl alcohol, chromatographically pure; Trifluoroacetic acid, analyzes 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 specificity experiment
Chromatographic condition: mobile phase: A: acetonitrile; B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH is to 3.0); A: B=425: 575 (V: V)
Sample solution is prepared: take each contamination levels product (A ~ H) and finished product respectively appropriate, accurately weighed, puts in same measuring bottle, adds thinning agent to suitable concn, shake up, and filtration, to obtain final product.Sample introduction also records chromatogram.
Experimental result: see accompanying drawing 1, main peak retention time is 12.3min, main peak and each impurity peaks good separation, and each impurity peaks all can baseline separation.
2) in bulk drug bendamustine hydrochloride, the quantitative limit of each impurity and detectability detect:
Chromatographic condition: mobile 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 is prepared: take each impurity reference substance respectively appropriate, accurately weighed, adds mobile phase to suitable concn, shakes up, and filters, to obtain final product.Sample introduction also records chromatogram.
In bulk drug bendamustine hydrochloride, the quantitative limit of each impurity the results are shown in following table:
In bulk drug bendamustine hydrochloride, the detectability of each impurity the results are shown in following table:
3) bendamustine hydrochloride determination of related substances-sample detection
Mobile phase: A: acetonitrile; B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH is to 3.0); A: B=425: 575 (V: V)
Sample solution is prepared: take finished product appropriate, accurately weighed, puts in same measuring bottle, adds mobile phase to suitable concn, shake up, obtain (1.0mg/ml).Sample introduction also records chromatogram.
Experimental result: see accompanying drawing 2, 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.
Embodiment 2 ~ 9: bendamustine hydrochloride determination of related substances (two ~ nine)
Mobile phase: A: acetonitrile; B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH);
Sample solution is prepared: take finished product appropriate, accurately weighed, puts in same measuring bottle, adds mobile phase to suitable concn, shake up, to obtain final product.Sample introduction also records 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.
Embodiment 10 ~ 13: bendamustine hydrochloride determination of related substances (ten ~ 13)
Instrument: Agilent 1200 type high performance liquid chromatograph (DAD detecting device)
Mobile phase: A: acetonitrile; B:0.02mol/l hexafluorophosphoric acid potassium solution (phosphoric acid adjust pH);
Sample solution is prepared: take finished product appropriate, accurately weighed, puts in same measuring bottle, adds mobile phase to suitable concn, shake up, to obtain final product.Sample introduction also records chromatogram.
Experimental result:
1) by accompanying drawing 11, extract ultraviolet spectrum and show the homolog that each impurity is finished product, uv-absorption maximum wavelength is identical.When determined wavelength is respectively 220nm, 233nm and 245nm, it is basically identical to there is level in each impurity.
2) by accompanying drawing 12, sample concentration is respectively 0.8,1.0,1.5mg/ml time; Each defects inspecting level is basically identical.
| Concentration | Impurity E | Unknown impuritie 1 | Unknown impuritie 2 |
| 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 bendamustine hydrochloride, described related substance is following impurity A-H:
Detection method is: sample solution testing sample mobile phase being mixed with 0.2mg/ml ~ 1.5mg/ml, and octadecylsilane chemically bonded silica chromatographic column carries out Reversed Phase Isocratic wash-out, and mobile phase is organic solvent and buffer solution; Described organic solvent is methyl alcohol or acetonitrile, and described buffer solution is the brine solution with acid for adjusting pH to 2.0 ~ 4.0; Described salt is hexafluorophosphate, and concentration is 0.01mol/L ~ 0.05mol/L.
2. analytical approach according to claim 1, described adjustment pH acid used is selected from phosphoric acid, acetic acid, formic acid or trifluoroacetic acid.
3. analytical approach according to claim 1, described salt is Potassium Hexafluorophosphate.
4. analytical approach according to claim 1, the volume ratio of described organic solvent in mobile phase is 40% ~ 45%.
5. analytical approach according to claim 1, testing conditions is: determined wavelength: 220 ~ 245nm; Mobile phase elution rate: 0.8 ~ 1.2ml/min; Chromatogram column temperature: 15 ~ 35 DEG C; Sample size: 20 μ l.
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| 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 |
| 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 |
| CN112394134B (en) * | 2019-08-14 | 2022-11-22 | 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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| HPLC study on the stability of bendamustine hydrochloride immobilized onto polyphosphoesters;Ivanka Penchenva等;《Journal of Pharmaceutical and Biomedical Analysis》;20080907;第48卷;1143-1150 * |
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| 盐酸苯达莫司汀(bendamustine hydrochloride);陈栋编译;《中国药物化学杂志》;20090430;第19卷(第2期);159-160 * |
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