CN1786706B - Cyclovirobuxine D raw medicine and method for determining its content in preparation by chromatography - Google Patents
Cyclovirobuxine D raw medicine and method for determining its content in preparation by chromatography Download PDFInfo
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- CN1786706B CN1786706B CN 200410093888 CN200410093888A CN1786706B CN 1786706 B CN1786706 B CN 1786706B CN 200410093888 CN200410093888 CN 200410093888 CN 200410093888 A CN200410093888 A CN 200410093888A CN 1786706 B CN1786706 B CN 1786706B
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- AXGWYABSYNCIMX-UHFFFAOYSA-N Cycloprotobuxin D Natural products C1CC(NC)C(C)(C)C2C31CC13CCC3(C)C(C(C)NC)CCC3(C)C1CC2 AXGWYABSYNCIMX-UHFFFAOYSA-N 0.000 title abstract 8
- GMNAPBAUIVITMI-UHFFFAOYSA-N Cyclovirobuxin D Natural products C1CC(NC)C(C)(C)C2C31CC13CCC3(C)C(C(C)NC)C(O)CC3(C)C1CC2 GMNAPBAUIVITMI-UHFFFAOYSA-N 0.000 title abstract 8
- 239000009642 cyclovirobuxine D Substances 0.000 title abstract 8
- 238000004587 chromatography analysis Methods 0.000 title 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 64
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- 229940079593 drug Drugs 0.000 claims abstract description 50
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 49
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- 238000013016 damping Methods 0.000 claims abstract description 40
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- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 31
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 31
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 52
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- 239000007788 liquid Substances 0.000 claims description 27
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 26
- 235000019253 formic acid Nutrition 0.000 claims description 26
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- 230000005526 G1 to G0 transition Effects 0.000 claims description 22
- 239000000377 silicon dioxide Substances 0.000 claims description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 17
- 239000000872 buffer Substances 0.000 claims description 16
- ADCGAPKUMAQOLJ-UHFFFAOYSA-N azane;formic acid Chemical compound N.OC=O.OC=O ADCGAPKUMAQOLJ-UHFFFAOYSA-N 0.000 claims 6
- OZZOVSQSDIWNIP-UHFFFAOYSA-N acetic acid;azane Chemical compound [NH4+].[NH4+].CC([O-])=O.CC([O-])=O OZZOVSQSDIWNIP-UHFFFAOYSA-N 0.000 claims 5
- 238000011095 buffer preparation Methods 0.000 claims 2
- 239000012467 final product Substances 0.000 claims 2
- 238000012360 testing method Methods 0.000 abstract description 60
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- YWEUIGNSBFLMFL-UHFFFAOYSA-N diphosphonate Chemical compound O=P(=O)OP(=O)=O YWEUIGNSBFLMFL-UHFFFAOYSA-N 0.000 description 16
- 238000001035 drying Methods 0.000 description 16
- DLYUQMMRRRQYAE-UHFFFAOYSA-N phosphorus pentoxide Inorganic materials O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 16
- 238000005303 weighing Methods 0.000 description 15
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- IDOHCEDWHOEHNL-UHFFFAOYSA-N Cyclovirobuxin C Natural products C1C23CCC4(C)C(C(C)N(C)C)C(O)CC4(C)C3CCC3C21CCC(NC)C3(C)C IDOHCEDWHOEHNL-UHFFFAOYSA-N 0.000 description 10
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- 239000007924 injection Substances 0.000 description 3
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- 238000013094 purity test Methods 0.000 description 3
- BDQNKCYCTYYMAA-UHFFFAOYSA-N 1-isocyanatonaphthalene Chemical compound C1=CC=C2C(N=C=O)=CC=CC2=C1 BDQNKCYCTYYMAA-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- DGTNSSLYPYDJGL-UHFFFAOYSA-N phenyl isocyanate Chemical compound O=C=NC1=CC=CC=C1 DGTNSSLYPYDJGL-UHFFFAOYSA-N 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
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- WWBITQUCWSFVNB-UHFFFAOYSA-N 3-silylpropan-1-amine Chemical group NCCC[SiH3] WWBITQUCWSFVNB-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a Cyclovirobuxine-D raw material drug and high efficiency liquid chromatography mensuration for Cyclovirobuxine-D content in different kinds of preparation, and high efficiency liquid chromatography for impurity testing in Cyclovirobuxine-D. The fixed phase in chromatographic condition uses octadecyl silane linkage silica gel or octyl silane linkage silica gel as filling material; The mobile phase adopt formate (ammonium formate) damping fluid-methanol system, acetic acid (ammonium acetate) damping fluid-methanol system, formate (ammonium formate) damping fluid-acetonitrile system or acetic acid (ammonium acetate) damping fluid-acetonitrile system; Evaporation light scattering detector (ELSD) detects drift tube temperature; drift tube temperature: 20-150 degree centigrade;and gas flow rate: 0.1L/min- 6.0L/min, split-flow or not split stream sampling. The method is easy to operate, timesaving, and has strong expert attribute. It could effectively divide some kind of alkaloids that has with Cyclovirobuxine-D in Cyclovirobuxine-D raw material drug. Thus, it is able to accurately measure their content. The method could also detecting impurity in Cyclovirobuxine-D. The invention could also measure content degree of homogeneity of Cyclovirobuxine-D in boxwood-nin piece, boxwood-nin drop pill, and boxwood-nin powder pin. And it commendably satisfies the request of pharmacopoeia content and content degree of homogeneity testing.
Description
Technical field
The present invention relates to the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content in cyclovimbuxine D bulk drug and the various preparation thereof (as Buxine Tablet, cyclovirobuxine dripping pills, buxenine powder pin etc.) and the cyclovimbuxine D.Particularly relate to the employing high performance liquid chromatography, and adopt evaporative light-scattering detector (ELSD) to detect.
Background technology
Buxaceae plant little leaf boxwood Buxus mincrophylla Sieb.et Zucc.var.sinica Rehd.et Wils. and belong to the complete stool plant together, through extracting and make with extra care the alkaloid of gained, if its major component cyclovimbuxine D content is greater than 70%, be the cyclovimbuxine D bulk drug, also be buxenine.
At present the assay method commonly used of the preparation of making to the raw material of cyclovimbuxine D or by the cyclovimbuxine D raw material is as follows:
Buxenine major component cyclovimbuxine D and other composition are alkaloid, so " 153 pages of contained non-aqueous titrations of Chinese pharmacopoeia version in 2000 are to measure alkaloidal classical way, this method is easy, but specificity is poor, promptly so long as alkaloid can both detect, so the result who obtains is alkaloidal total alkali content, it is the total amount of cyclovimbuxine D and other composition, and cyclovimbuxine D is a kind of alkaloid wherein, adopts this detection method can not accurately measure the real content of cyclovimbuxine D.
With buxenine is the preparation Buxine Tablet that former medicine is produced, " 575 pages of contained methods of Chinese pharmacopoeia version in 2000 are acid-dye colorimetries for it, this method complex operation, and specificity is poor, promptly so long as alkaloid can both detect, so the result who obtains is alkaloidal total alkali content, i.e. the total amount of cyclovimbuxine D and its related substance, and cyclovimbuxine D is a kind of alkaloid wherein, adopts this detection method can not accurately measure the real content of cyclovimbuxine D.
With buxenine is that preparation cyclovirobuxine dripping pills, the buxenine powder pin of former medicine production also adopts acid-dye colorimetry to measure the content of its contained cyclovimbuxine D at present, and what promptly record also is the alkaloid total alkali content, can not accurately measure the wherein content of cyclovimbuxine D.
Because there is the shortcoming of specificity difference in the said determination method, so many scientific research personnel actively seek the strong assay method of specificity, high-efficient liquid phase technique particularly, as pre-column derivatization high performance liquid chromatography, terminal absorption high performance liquid chromatography, high performance liquid chromatogram-mass spectroscopy etc., but all there is such-and-such shortcoming in these methods.
Adopt the content of pre-column derivatization high effective liquid chromatography for measuring cyclovimbuxine D, need before sample introduction, make cyclovimbuxine D generate new compound by chemical reaction, be converted to the content of cyclovimbuxine D again by the content of measuring this new compound, so be indirect determination method, the method complex operation, and derivatization reagent all adopts phenyl isocyanate or 1-naphthyl isocyanate, phenyl isocyanate and 1-naphthyl isocyanate are the liquid that is harmful to, be used as dacryagogue, it has pungency, photonasty, to light and wet responsive, storage is preserved after must filling nitrogen, the conventional preservation easily lost efficacy, and long-term the use has very big injury to human body, so be not suitable for being used as the content that derivatization reagent is measured cyclovimbuxine D.
Application number: 200310106142.8, publication number: the patent of invention of CN 1540339A adopts the terminal content that absorbs the high effective liquid chromatography for measuring cyclovimbuxine D, is filling agent but the method adopts aminopropyl silane group silica gel, and versatility is not strong; And what use is that the terminal 210nm of absorption detects, and selectivity is not strong, so when the cyclovimbuxine D in Buxine Tablet, cyclovirobuxine dripping pills and the buxenine powder pin measured, the pre-treatment complex operation.
Also have document to adopt high performance liquid chromatogram-mass spectroscopy in addition, this method precision is relatively poor, and expense is more expensive, and people do not adopt the assay method of this method as cyclovimbuxine D substantially at present.
Summary of the invention
It is strong to the purpose of this invention is to provide a kind of specificity; Simple to operate, save time; Toxic and side effect is little; Highly versatile; The high-performance liquid chromatogram determination method of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug that applicability is wide and the various preparation thereof.
The present invention has overcome " Chinese pharmacopoeia contained existing shortcoming of non-aqueous titration of version one one 153 pages mensuration cyclovimbuxine D in 2000, has the strong advantage of specificity, when measuring, can accurately measure the real content of cyclovimbuxine D, and can measure related substance.
The present invention has overcome " the existing shortcoming of acid-dye colorimetry of 575 pages of contained Buxine Tablets of Chinese pharmacopoeia version in 2000, has the strong advantage of specificity, when measuring, can accurately measure the real content of cyclovimbuxine D, and can measure related substance, and method of operating of the present invention is easy.
The present invention has also overcome present employing acid-dye colorimetry and has measured the shortcoming that the content of cyclovirobuxine dripping pills, the contained cyclovimbuxine D of buxenine powder pin exists, has the strong advantage of specificity, when measuring, can accurately measure the real content of cyclovimbuxine D, and can measure related substance, and method of operating of the present invention is easy.
Assay method of the present invention has overcome the existing shortcoming that adopts pre-column derivatization high effective liquid chromatography for measuring cyclovimbuxine D, adopt the direct injected method to measure the content of cyclovimbuxine D, method is simple, and does not need to use human body is had derivatization reagent than major injury.
The present invention has also overcome application number: 200310106141.3, publication number: the patent of invention of CN 1540338A adopts the terminal shortcoming that absorbs the content of high effective liquid chromatography for measuring cyclovimbuxine D, it is filling agent that the present invention adopts octadecylsilane chemically bonded silica commonly used or octyl silane group silica gel, highly versatile; And the present invention adopts the direct sample dissolution sample introduction of moving phase, and is simple to operate.
Purpose of the present invention can realize by following measures:
Adopt high performance liquid chromatograph, stationary phase employing octadecylsilane chemically bonded silica or octyl silane group silica gel are filling agent in the chromatographic condition; Moving phase adopts formic acid (ammonium formate) damping fluid-methanol system, acetate (ammonium acetate) damping fluid-methanol system, formic acid (ammonium formate) damping fluid-acetonitrile system or acetate (ammonium acetate) damping fluid-acetonitrile system; Adopt evaporative light-scattering detector (ELSD) to detect drift tube temperature: 20～150 ℃; Gas flow rate: 0.1L/min～6.0L/min.
In the high performance liquid chromatography of determination of foreign matter, it is filling agent that stationary phase adopts octadecylsilane chemically bonded silica or octyl silane group silica gel in the high performance liquid chromatography of cyclovimbuxine D content of the present invention and the cyclovimbuxine D; Moving phase adopts formic acid (ammonium formate) damping fluid-methanol system, acetate (ammonium acetate) damping fluid-methanol system, formic acid (ammonium formate) damping fluid-acetonitrile system.
In the high performance liquid chromatography of determination of foreign matter, it is filling agent that stationary phase adopts octadecylsilane chemically bonded silica in the high performance liquid chromatography of best cyclovimbuxine D content of the present invention and the cyclovimbuxine D; Moving phase adopts formic acid (ammonium formate) damping fluid-methanol system, acetate (ammonium acetate) damping fluid-methanol system.
Moving phase formic acid of the present invention (ammonium formate) damping fluid-methanol system, acetate (ammonium acetate) damping fluid-methanol system adopts following method configuration moving phase: the ammonium formate damping fluid (is got formic acid 0.5～50ml, add about water 800ml, add ammonium formate adjust pH to 2.3～6.0, add water to 1000ml)-(volume ratio is 35～89: 65～11) (get acetate 0.5～50ml for moving phase or ammonium acetate buffer for methyl alcohol or acetonitrile, add about water 800ml, add ammonium acetate adjust pH to 2.3～6.0, add water to 1000ml)-(volume ratio is 35～89: 65～11) be moving phase for methyl alcohol or acetonitrile.
Preferred moving phase formic acid of the present invention (ammonium formate) damping fluid-methanol system adopts following method configuration moving phase: the ammonium formate damping fluid (is got formic acid 2～35ml, add about water 800ml, add ammonium formate adjust pH to 2.7～5.5, add water to 1000ml)-(volume ratio is 50～74: 50～26) (get formic acid 2～35ml for moving phase or ammonium acetate buffer for methyl alcohol or acetonitrile, add about water 800ml, add ammonium acetate adjust pH to 2.7～5.5, add water to 1000ml)-(volume ratio is 50～74: 50～26) be moving phase for methyl alcohol or acetonitrile.
Best moving phase formic acid of the present invention (ammonium formate) damping fluid-methanol system adopts following method configuration moving phase: the ammonium formate damping fluid (is got formic acid 5～20ml, add about water 800ml, add ammonium formate adjust pH to 3.0～5.0, add water to 1000ml)-(volume ratio is 55～69: 45～31) (get acetate 5～20ml for moving phase or ammonium acetate buffer for methyl alcohol or acetonitrile, add about water 800ml, add ammonium acetate adjust pH to 3.0～5.0, add water to 1000ml)-(volume ratio is 55～69: 45～31) be moving phase for methyl alcohol or acetonitrile.
The configuration of the moving phase damping fluid described in the present invention, the reagent of getting, test solution and the proportional increase of water or minimizing and configuration sequence change, and do not influence protection domain of the present invention.
In the high performance liquid chromatography of determination of foreign matter, adopting the split sampling drift tube temperature is 20～100 ℃, gas flow rate: 0.1L/min～4.0L/min in the high performance liquid chromatography of cyclovimbuxine D content of the present invention and the cyclovimbuxine D.
In the high performance liquid chromatography of preferred cyclovimbuxine D content of the present invention and the cyclovimbuxine D in the high performance liquid chromatography of determination of foreign matter, adopting the split sampling drift tube temperature is 30～80 ℃, gas flow rate: 0.3L/min～3.0L/min.
In the high performance liquid chromatography of best of the present invention bad virobuxine D content and the cyclovimbuxine D in the high performance liquid chromatography of determination of foreign matter, adopting the split sampling drift tube temperature is 40～70 ℃, gas flow rate: 0.5L/min～2.0L/min.
In the high performance liquid chromatography of cyclovimbuxine D content of the present invention and the cyclovimbuxine D in the high performance liquid chromatography of determination of foreign matter, adopt not that the split sampling drift tube temperature is 70～150 ℃, gas flow rate: 0.1L/min～6.0L/min.
In the high performance liquid chromatography of preferred cyclovimbuxine D content of the present invention and the cyclovimbuxine D in the high performance liquid chromatography of determination of foreign matter, adopt not that the split sampling drift tube temperature is 80～140 ℃, gas flow rate: 1.0L/min～4.5L/min.
In the high performance liquid chromatography of best cyclovimbuxine D content of the present invention and the cyclovimbuxine D in the high performance liquid chromatography of determination of foreign matter, adopt not that the split sampling drift tube temperature is 90～120 ℃, gas flow rate: 1.5L/min～3.5L/min.
In the high performance liquid chromatography of determination of foreign matter, cyclovimbuxine D bulk drug and various preparation thereof directly with the moving phase dissolving, filter, and advance high performance liquid chromatograph in the high performance liquid chromatography of cyclovimbuxine D content of the present invention and the cyclovimbuxine D.
The preparation made from the cyclovimbuxine D bulk drug described in the present invention can be made any form on the pharmacy, comprises injection and oral formulations.Wherein injection comprises parenteral solution, drip solution, powder-injection; Oral formulations comprises granule, tablet, electuary, powder, oral liquid, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, sucks agent, granule, pill, paste, sublimed preparation, spray, pill, disintegrant, oral disintegrating tablet, micropill etc.
Advantage of the present invention:
The present invention adopts high-efficient liquid phase chromatogram technology, and evaporative light-scattering detector (ELSD) detects, and measures the content of cyclovimbuxine D in cyclovimbuxine D bulk drug and the various preparation (as Buxine Tablet, cyclovirobuxine dripping pills, buxenine powder pin etc.) thereof.Specificity is strong; Simple to operate, save time; Toxic and side effect is little; Highly versatile; The high-performance liquid chromatogram determination method of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug that applicability is wide and the various preparation thereof.
Description of drawings
Provide cyclovimbuxine D bulk drug chromatogram, cyclovimbuxine D reference substance purity test figure, refining cyclovimbuxine D material purity inspection chromatogram, cyclovirobuxine dripping pills assay chromatogram, Buxine Tablet agent content mensuration chromatogram, buxenine powder pin assay chromatogram below, be intended to further specify the present invention, but be not that the present invention is limited.
Figure one is a cyclovimbuxine D bulk drug chromatogram
Figure two is cyclovimbuxine D reference substance purity test figure
Figure three is refining cyclovimbuxine D material purity controlling chart
Figure four is a cyclovirobuxine dripping pills assay chromatogram
Figure five measures chromatogram for the Buxine Tablet agent content
Figure six is a buxenine powder pin assay chromatogram
Embodiment
The invention will be further described by the following examples.
Chromatographic column adopting Di Ma company product [Diamonsil (diamond) C18 5 μ m, 250 * 4.6mm]
Embodiment one
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get ammonium formate 15.0g, add formic acid 10ml, add water and make dissolving and be diluted to 1000ml)-methyl alcohol (volume ratio is 62: 38) is moving phase; Flow velocity is 0.8ml/min; Evaporative light-scattering detector: drift tube temperature is 100 ℃, and gas flow rate is 2.7L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Accurate respectively reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, sample size (μ g) according to the peak area of chromatogram and sample, reference substance, calculate with the external standard two-point method, that is, computing formula is as follows:
Reference substance regression equation: Y=k * X+b (k, b are constant)
Sample size: C=100% * [25 * (Y-b)]/(20 * k * W)
Y: the peak area value of sample
W: the sample weighting amount of sample (mg)
C: the content of cyclovimbuxine D (%) test sample chromatogram is seen figure one in the cyclovimbuxine D bulk drug, and the result shows that content is 91.12%.
Embodiment two (cyclovimbuxine D reference substance purity test)
Cyclovimbuxine D reference substance (checking institute, lot number 888-200001) available from Chinese pharmaceutical biological product
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get ammonium formate 15.0g, add formic acid 10ml, add water and make dissolving and be diluted to 1000ml)-methyl alcohol (volume ratio is 62: 38) is moving phase; Flow velocity is 0.8ml/min; Evaporative light-scattering detector: drift tube temperature is 100 ℃, and gas flow rate is 2.7L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
Cyclovimbuxine D reference substance 10mg is got in the preparation of need testing solution, accurate claims surely, puts in the 5ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, as cyclovimbuxine D reference substance enriched sample H; The accurate 1ml that draws puts in the 10ml measuring bottle, adds moving phase and is diluted to scale, shakes up, as cyclovimbuxine D reference substance low concentration sample L.
Investigate specificity, precision and the linearity of sample L, the result is all good.
Adopt Self-control method that the related substance of above-mentioned sample is detected:
Get above-mentioned sample H and each 20ul of L, inject high performance liquid chromatograph respectively, be calculated as follows the content of relative substance: the peak area of the impurity %=100% in the sample * (all the impurity peak area sums in the H chromatogram except that main peak) * 10 ÷ L chromatogram main peaks, the impurity content that calculates in the cyclovimbuxine D reference substance is 3.93%.The H chromatogram is seen accompanying drawing two.
Embodiment three (refining cyclovimbuxine D material purity is checked)
Refining cyclovimbuxine D raw material (company limited provides by the technological development of the Kang Li of Henan Province medicine)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get ammonium formate 15.0g, add formic acid 10ml, add water and make dissolving and be diluted to 1000ml)-methyl alcohol (volume ratio is 63: 37) is moving phase; Flow velocity is 0.8ml/min; Evaporative light-scattering detector: drift tube temperature is 100 ℃, and gas flow rate is 2.7L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
Refining cyclovimbuxine D raw material 10mg is got in the preparation of need testing solution, accurately claims surely, puts in the 2ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, as refining cyclovimbuxine D raw material enriched sample H1; The accurate 1ml that draws puts in the 100ml measuring bottle, adds moving phase and is diluted to scale, shakes up, as refining cyclovimbuxine D raw material low concentration sample L1.
Investigate specificity, precision and the linearity of sample L1, the result is all good.
Adopt Self-control method that the related substance of above-mentioned sample is detected:
Get above-mentioned sample H1 and each 20ul of L1, inject high performance liquid chromatograph respectively, be calculated as follows the content of relative substance: the peak area of the impurity %=100% in the sample * (all the impurity peak area sums in the H1 chromatogram except that main peak) ÷ L1 chromatogram main peak, calculating refining cyclovimbuxine D impurities in raw materials content is 0.38%.The H1 chromatogram is seen accompanying drawing three.
Embodiment four
Cyclovirobuxine dripping pills (providing) by Tianjin Tasly Pharmaceutical Co., Ltd
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get ammonium formate 15.0g, add formic acid 10ml, add water and make dissolving and be diluted to 1000ml)-methyl alcohol (volume ratio is 60: 40) is moving phase; Flow velocity is 0.8ml/min; Evaporative light-scattering detector: drift tube temperature is 100 ℃, and gas flow rate is 2.7L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 4mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
20 of cyclovirobuxine dripping pills are got in the preparation of need testing solution, and accurate the title decides, porphyrize, and precision takes by weighing in right amount (being equivalent to cyclovimbuxine D 1.5mg approximately), put in the 10ml volumetric flask, it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shake up, cross 0.45 μ m miillpore filter, promptly.
Accurate respectively reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, the test sample chromatogram is seen figure four, measurement ring virobuxine D peak area is pressed the external standard two-point method with calculated by peak area content, and the result shows that content is 95.1% of labelled amount.
Uniformity of dosage units is got 10 of cyclovirobuxine dripping pills, puts respectively in the 5ml measuring bottle, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, need testing solution.Measure according to the method under the assay item, press the external standard two-point method, get every content, more promptly get uniformity of dosage units with calculated by peak area content.
Embodiment five
Buxine Tablet (commercially available, company limited produces by Chinese Shaanxi Hai Mupu medicine made in Germany)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; (volume ratio is to be moving phase at 62: 38 to ammonium acetate buffer (get ammonium acetate 15.0g, add acetate 10ml, add water and make dissolving and be diluted to 1000ml)-methyl alcohol; Flow velocity is 0.8ml/min; Evaporative light-scattering detector: drift tube temperature is 100 ℃, and gas flow rate is 2.7L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 4mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
20 of Buxine Tablets are got in the preparation of need testing solution, and accurate the title decides, porphyrize, and precision takes by weighing in right amount (being equivalent to cyclovimbuxine D 1.5mg approximately), put in the 10ml volumetric flask, it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shake up, cross 0.45 μ m miillpore filter, promptly.
Accurate respectively reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the test sample chromatogram is seen figure five, and measurement ring virobuxine D peak area is pressed the external standard two-point method with calculated by peak area content.
Uniformity of dosage units is got 10 of Buxine Tablets, puts respectively in the 5ml measuring bottle, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, need testing solution.Measure according to the method under the assay item, press the external standard two-point method, get every content, more promptly get uniformity of dosage units with calculated by peak area content.
Embodiment six
Buxenine powder pin (company limited provides by the technological development of the Kang Li of Henan Province medicine)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get ammonium formate 15.0g, add formic acid 10ml, add water and make dissolving and be diluted to 1000ml)-methyl alcohol (volume ratio is 62: 38) is moving phase; Flow velocity is 0.8ml/min; Evaporative light-scattering detector: drift tube temperature is 100 ℃, and gas flow rate is 2.7L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 4mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
Buxenine powder pin an amount of (being equivalent to cyclovimbuxine D 1.5mg approximately) is got in the preparation of need testing solution, accurately claims surely, puts in the 10ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Accurate respectively reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the test sample chromatogram is seen figure six, and measurement ring virobuxine D peak area is pressed the external standard two-point method with calculated by peak area content.
Following examples seven～embodiment ten all adopts Di Ma company chromatographic column: Kromasil C8 5 μ m, 250 * 4.6mm.
Embodiment seven
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase are filling agent with octyl silane group silica gel; Ammonium formate damping fluid (get ammonium formate 15.0g, add formic acid 10ml, add water and make dissolving and be diluted to 1000ml)-methyl alcohol (volume ratio is 62: 38) is moving phase; Flow velocity is 0.8ml/min; The SEDEX evaporative light-scattering detector: drift tube temperature is 35 ℃, and gas flow rate is 1.0L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Accurate respectively reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, sample size (μ g) according to the peak area of chromatogram and sample, reference substance, calculate with the external standard two-point method, that is, computing formula is as follows:
Reference substance regression equation: Y=k * X+b (k, b are constant)
Sample size: C=100% * [25 * (Y-b)]/(20 * k * W)
Y: the peak area value of sample
W: the sample weighting amount of sample (mg)
C: the content of cyclovimbuxine D (%) in the cyclovimbuxine D bulk drug
Embodiment eight
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase are filling agent with octyl silane group silica gel; Ammonium acetate buffer (get ammonium acetate 18.0g, add acetate 10ml, add water and make dissolving and be diluted to 1000ml)-methyl alcohol (volume ratio is 62: 38) is moving phase; Flow velocity is 0.8ml/min; The SEDEX evaporative light-scattering detector: drift tube temperature is 75 ℃, and gas flow rate is 2.0L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Embodiment nine
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase are filling agent with octyl silane group silica gel; Ammonium acetate buffer (get ammonium acetate 18.0g, add acetate 10ml, add water and make dissolving and be diluted to 1000ml)-acetonitrile (volume ratio is 68: 32) is moving phase; Flow velocity is 0.8ml/min; Evaporative light-scattering detector: drift tube temperature is 90 ℃, and gas flow rate is 2.0L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Embodiment ten
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase are filling agent with octyl silane group silica gel; Ammonium formate damping fluid (get ammonium formate 15.0g, add formic acid 8ml, add water and make dissolving and be diluted to 1000ml)-acetonitrile (volume ratio is 68: 32) is moving phase; Flow velocity is 0.8ml/min; The SEDEX evaporative light-scattering detector: drift tube temperature is 50 ℃, and gas flow rate is 1.5L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Following examples 11～embodiment 14 all adopts Di Ma company product [Diamonsil (diamond) C185 μ m, 250 * 4.6mm]
Embodiment 11
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium acetate buffer (get acetate 2ml, add about water 800ml, with ammonium acetate adjust pH to 5.5, add water and make dissolving and be diluted to 1000ml)-methyl alcohol or acetonitrile (volume ratio is 50: 50) are moving phase; Flow velocity is 0.5ml/min; Evaporative light-scattering detector: drift tube temperature is 80 ℃, and gas flow rate is 1.5L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Embodiment 12
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium acetate buffer (get acetate 35ml, add about water 800ml, with ammonium acetate adjust pH to 2.7, add water and make dissolving and be diluted to 1000ml)-methyl alcohol or acetonitrile (volume ratio is 74: 26) are moving phase; Flow velocity is 0.5ml/min; Evaporative light-scattering detector: drift tube temperature is 80 ℃, and gas flow rate is 1.5L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Embodiment 13
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get formic acid 5ml, add about water 800ml, with ammonium formate adjust pH to 5.0, add water and make dissolving and be diluted to 1000ml)-methyl alcohol or acetonitrile (volume ratio is 55: 45) are moving phase; Flow velocity is 1.0ml/min; Evaporative light-scattering detector: drift tube temperature is 120 ℃, and gas flow rate is 3.5L/min; Column temperature is 40 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Embodiment 14
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get formic acid 20ml, add about water 800ml, with ammonium formate adjust pH to 3.0, add water and make dissolving and be diluted to 1000ml)-methyl alcohol or acetonitrile (volume ratio is 69: 31) are moving phase; Flow velocity is 1.0ml/min; Evaporative light-scattering detector: drift tube temperature is 120 ℃, and gas flow rate is 3.5L/min; Column temperature is 40 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium acetate buffer (get acetate 2ml, add about water 800ml, with ammonium acetate adjust pH to 5.5, add water and make dissolving and be diluted to 1000ml)-methyl alcohol or acetonitrile (volume ratio is 50: 50) are moving phase; Flow velocity is 0.5ml/min; Evaporative light-scattering detector: split sampling, drift tube temperature are 30 ℃, and gas flow rate is 0.8L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Embodiment 16
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium acetate buffer (get acetate 35ml, add about water 800ml, with ammonium acetate adjust pH to 2.7, add water and make dissolving and be diluted to 1000ml)-methyl alcohol or acetonitrile (volume ratio is 74: 26) are moving phase; Flow velocity is 0.5ml/min; Evaporative light-scattering detector: split sampling, drift tube temperature are 30 ℃, and gas flow rate is 0.8L/min; Column temperature is 30 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Embodiment 17
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get formic acid 5ml, add about water 800ml, with ammonium formate adjust pH to 5.0, add water and make dissolving and be diluted to 1000ml)-methyl alcohol or acetonitrile (volume ratio is 55: 45) are moving phase; Flow velocity is 1.0ml/min; Evaporative light-scattering detector: split sampling, drift tube temperature are 80 ℃, and gas flow rate is 2.0L/min; Column temperature is 40 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Embodiment 18
Cyclovimbuxine D bulk drug commercial (effluent south dragon all pharmaceutical factory produces)
Chromatographic condition and system flexibility stationary phase octadecylsilane chemically bonded silica are filling agent; Ammonium formate damping fluid (get formic acid 20ml, add about water 800ml, with ammonium formate adjust pH to 3.0, add water and make dissolving and be diluted to 1000ml)-methyl alcohol or acetonitrile (volume ratio is 69: 31) are moving phase; Flow velocity is 1.0ml/min; Evaporative light-scattering detector: split sampling, drift tube temperature are 80 ℃, and gas flow rate is 2.0L/min; Column temperature is 40 ℃; In the need testing solution chromatogram, cyclovimbuxine D peak and other alkaloid peak all should reach baseline separation; Theoretical cam curve is pressed the cyclovimbuxine D peak and is calculated, and should be not less than 2000.
It is drying agent that the preparation of reference substance solution is learnt from else's experience with phosphorus pentoxide, and 60 ℃ of constant temperature drying under reduced pressure are to the cyclovimbuxine D reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing cyclovimbuxine D bulk drug 9mg, puts in the 25ml volumetric flask, and it is an amount of to add moving phase, ultrasonicly makes dissolving, and is diluted to scale, shakes up, and crosses 0.45 μ m miillpore filter, promptly.
Respectively accurate reference substance solution 10 μ l, the 20 μ l of drawing of determination method, need testing solution 20 μ l inject high performance liquid chromatograph, and the sample size (μ g) according to the peak area of chromatogram and sample, reference substance calculates with the external standard two-point method, promptly.
Claims (9)
1. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in a cyclovimbuxine D bulk drug and the various preparation thereof, it is characterized in that its chromatographic condition is: chromatographic column is that stationary phase employing octadecylsilane chemically bonded silica or octyl silane group silica gel are filling agent, and column temperature is 30-40 ℃; Moving phase adopts formic acid-ammonium formate damping fluid: methanol system, acetate-ammonium acetate buffer: methanol system, formic acid-ammonium formate damping fluid: acetonitrile system or acetate-ammonium acetate buffer: acetonitrile system, it is 50～74: 50～26 that described moving phase is formed volume ratio, flow velocity is 0.5-1.0ml/min, wherein ammonium formate pH value is 3.0-5.0, and ammonium acetate buffer pH value is 2.7-5.5; Adopt ELSD type evaporative light-scattering detector, not its drift tube temperature during split sampling: 80～120 ℃, gas flow rate: 1.5L/min～3.5L/min, its drift tube temperature during split sampling: 30～80 ℃, gas flow rate: 0.8L/min～2.0L/min; Described detecting device can also be SEDEX type evaporative light-scattering detector, and its drift tube temperature is 35-75 ℃; Gas flow rate: 1.0L/min～2.0L/min.
2. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug according to claim 1 and the various preparation thereof is characterized in that moving phase adopts formic acid-ammonium formate damping fluid in its chromatographic condition: methanol system, acetate-ammonium acetate buffer: methanol system or formic acid-ammonium formate damping fluid: acetonitrile system.
3. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug according to claim 2 and the various preparation thereof is characterized in that moving phase adopts formic acid-ammonium formate damping fluid in its chromatographic condition: methanol system or acetate-ammonium acetate buffer: methanol system.
4. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug according to claim 1 and the various preparation thereof, it is characterized in that, described formic acid-ammonium formate damping fluid is prepared according to following method: get formic acid 0.5～50ml, add water 800ml, add ammonium formate adjust pH to 3.0～5.0, add water to 1000ml and get final product; Described acetate-ammonium acetate buffer is prepared according to following method: get acetate 0.5～50ml, add water 800ml, add ammonium acetate adjust pH to 2.7～5.5, add water to 1000ml and get final product.
5. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug according to claim 4 and the various preparation thereof, it is characterized in that, in the described ammonium formate damping fluid preparation: get formic acid 2～35ml; In the described ammonium acetate buffer preparation: get acetate 2～35ml.
6. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug according to claim 5 and the various preparation thereof, it is characterized in that, in the described ammonium formate damping fluid preparation: get formic acid 5～20ml; In the described ammonium acetate buffer preparation: get acetate 5～20ml.
7. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug according to claim 1 and the various preparation thereof is characterized in that: it is 55～69: 45～31 that described moving phase is formed volume ratio.
8. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug according to claim 1 and the various preparation thereof, the split sampling drift tube temperature is 90～120 ℃ to its feature adopting not, gas flow rate: 2.0L/min～3.5L/min.
9. the high performance liquid chromatography of determination of foreign matter in the high performance liquid chromatography of cyclovimbuxine D content and the cyclovimbuxine D in cyclovimbuxine D bulk drug according to claim 1 and the various preparation thereof, it is characterized in that cyclovimbuxine D bulk drug and various preparation thereof directly dissolve with moving phase, filter, advance high performance liquid chromatograph.
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| CN101191787B (en) * | 2006-11-21 | 2011-07-27 | 上海医药工业研究院 | High performance liquid chromatography method for measuring doripenem content |
| CN101135676B (en) * | 2007-07-23 | 2010-08-11 | 江苏省原子医学研究所 | Boc protected thymidine derivates BOC-FLT content analysis method |
| CN101126747B (en) * | 2007-08-16 | 2010-08-11 | 江苏省原子医学研究所 | Tumour cell proliferation imaging agent precursor content analysis method |
| CN102590378B (en) * | 2012-02-05 | 2013-09-11 | 西北农林科技大学 | Method for detecting content of swainsonine in locoweed endophytic fungi |
| CN106353154B (en) * | 2016-08-08 | 2019-01-01 | 杭州民生药业有限公司 | The method for separating and preparing of a four peptides impurity in alanyl-glutamine injection |
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