CN104569170A

CN104569170A - Method for detecting ginkgo leaf extract and diphyridamole injection preparation

Info

Publication number: CN104569170A
Application number: CN201310521033.6A
Authority: CN
Inventors: 窦啟玲; 杨青波; 陈伟; 陆煜玫; 刘秋
Original assignee: Guizhou Yibai Pharmaceutical Co Ltd
Current assignee: Guizhou Yibai Pharmaceutical Co Ltd
Priority date: 2013-10-29
Filing date: 2013-10-29
Publication date: 2015-04-29
Anticipated expiration: 2033-10-29
Also published as: CN104569170B

Abstract

The invention provides a method for detecting a ginkgo leaf extract and diphyridamole injection preparation. The method comprises the step of fingerprint test and content test of the ginkgo leaf extract and diphyridamole injection preparation, wherein the fingerprint test refers to fingerprint test of flavonoid and terpene lactones in a gingko leaf extract; and the content test refers to test of flavonoid and terpene lactones in the gingko leaf extract. Compared with the prior art, the method can realize more effective product quality control, as well as authentication and content test of effective component monomers, of the ginkgo leaf extract and diphyridamole injection preparation, is very good in precision, stability and repeatability and is another excellent guarantee for the variety curative effect.

Description

A kind of detection method of ginkgo-dipyridamine injection

Technical field:

The present invention relates to a kind of detection method of ginkgo-dipyridamine injection, belong to technical field of pharmaceuticals.

Technical background:

Cardiovascular and cerebrovascular disease, as the most common in coronary heart disease, cerebral thrombus, hypertension, the cerebral infarction Deng Junshi world today and endanger one of maximum disease, be also common disease, the frequently-occurring disease of harm our people health, become the one of the main reasons of human mortality; It was reported, the incidence of disease in recent years has and increases trend year by year, and Er Qiezhong, adolescent patients constantly increase.Ginkgo Damo injection is made by ginkgo biloba p.e and Dipyridamole, records in chemicals provincial standard rising national standard (first), standard No.: WS-10001-(HD-0087)-2002.This product energy platelet aggregation-against, belong to coronary artery expansion medicine, be applicable to prevention and therapy coronary heart disease, thrombotic disease, wherein contain flavonoids, terpene, phenols, alkaloid isoreactivity composition in ginkgo biloba p.e, can play and improve cardiac muscle and brain tissue metabolism, increase coronary artery and brain vessel blood, improve the effect of the blood confessions such as cardiac muscle and brain tissue, good curative effect is had for treatment cardiovascular and cerebrovascular disease such as coronary heart diseases and angina pectoris, arrhythmia cordis, cerebral thrombus, senile dementia etc., can improve immunity of organisms, resist the disease occurs simultaneously.But pharmaceutical preparation must on the basis ensureing constant product quality controllable safety, more new development that could not be short.

Document " dual qualitative and dual quantitative similarity method evaluates the efficient liquid-phase chromatograph finger print atlas of Ginkgo Damo injection " (Sun Guoxiang etc., chromatogram the 25th volume the 4th phase, in July, 2007) disclose the HPLC finger-print evaluating Ginkgo Damo injection with double qualitative similarities S and S, double quantitative similarities C and P in a literary composition, with Kaempferol, rutin, Quercetin, Isorhamnetin, Ginkgolides a and B and C for reference substance, wherein chromatographic condition is CenturySIL C18BDS post, mobile phase A: water (containing 1% acetic acid); B: acetonitrile (containing 1% acetic acid).Gradient elution, order is 0-20min, 0%-15%B; 20-35min, 15%-22%B; 35-45min, 22%-26%B; 45-55min, 26%-32%B; 55-70min, 32%-55%B; 70-80min, 55%-62%B.Flow velocity 1.0mL/min, determined wavelength 265nm, column temperature 30.0 ± 0.15 DEG C, elution time 80min.Although this detection method can control the quality of Ginkgo Damo injection preferably, assay method is complicated, and as also incomplete in the separation, evaluation etc. of Bilobalide etc. to other concrete effective constituents of product.Document " research of Ginkgo Damo injection finger-print " (Song Jingyan, scientific and technological information exploitation and economic 18th volume the 6th phase, in July, 2008) disclose finger-print research to ginkgo biloba p.e and injection in a literary composition, take rutin as reference substance, wherein chromatographic condition is octadecylsilane chemically bonded silica is filling agent, mobile phase A: acetonitrile; B:0.4% phosphoric acid solution (regulating pH to 2.5 with triethylamine).Gradient elution, order is 0-30min, 85%-83%B; 30-40min, 83%-80%B; 40-60min, 80%-75%B; 60-80min, 75%-85%B.Flow velocity 1.0mL/min, determined wavelength 360nm, column temperature 40.0 DEG C, elution time 80min.The method only with rutin in contrast, can not well be demarcated terpene lactones such as flavonoids and ginkalide A such as the Quercetins in ginkgo biloba p.e and make a comment or criticism, also cannot evaluate the content of its effective constituent.In order to better control the quality of said preparation, ensure the security of medication, better Instructing manufacture, make technology controlling and process more rationally strict, make consumer's energy full appreciation product quality, need constantly to study and find out the method being more conducive to product quality detection, clear and definite specifically effective constituent kind and content.

Summary of the invention:

The object of this invention is to provide a kind of detection method of ginkgo-dipyridamine injection, this method provides the index of detection, the means, technical method etc. of detection for following product to relevant production, testing agency, better to control the quality of said preparation, ensure the security of medication, can better Instructing manufacture, make controlling of production process more strict rationally, make consumer can full appreciation product quality, for product be:

The injection be prepared from by ginkgo biloba p.e 20-100 weight portion, Dipyridamole 2-10 parts by weight of composition, comprise be directly used in drug administration by injection parenteral solution, need dilute after for the concentrated solution for injection of drip-feed, the injection sterile powder directly obtained for the glucose intravenous infusion of drip-feed and sodium chloride intravenous infusion and freeze-drying or spray drying process and aseptic block.

Such as, described product can be commercially available Ginkgo Damo injection, and specification is 5ml, 10ml, is produced by 3 companies such as Guizhou benefit one hundred pharmacy, the general moral medicine company in Shanxi, the red pharmacy of Tonghua paddy; Also can be patent CN200510200678.5, name is called: freeze-dried formulation prepared by the preparation method that " ginkgo Damo freeze-drying preparation and preparation method thereof " provides, also can be patent CN200810068921.6, name be called: ejection preparation prepared by the preparation method that " composite preparation of ginkgo biloba p.e and Dipyridamole and preparation method thereof " provides.

The present invention is formed like this:

The detection method of ginkgo-dipyridamine injection, comprises following content:

The finger-print test of ginkgo-dipyridamine injection, comprises and is characterized as main finger-print with ginkgo biloba p.e flavones ingredient and based on the finger-print of ginkgo biloba p.e terpene lactone composition characteristics;

The assay of ginkgo-dipyridamine injection, comprises and is characterized as main assay with ginkgo biloba p.e flavones ingredient, is embodied in the content of Quercetin, Kaempferol, Isorhamnetin; With based on the assay of ginkgo biloba p.e terpene lactone composition characteristics, be embodied in the content of Ginkgolide A. B. C and Bilobalide.

Wherein, fingerprint atlas detection method comprises following content:

A, the test of employing high performance liquid chromatography are characterized as main finger-print with ginkgo biloba p.e flavones ingredient:

(1) preparation of need testing solution: get ginkgo-dipyridamine injection to be measured appropriate, parenteral solution volatilizes moisture, and freeze-dried powder is directly placed in the mixed liquor of methyl alcohol-25% hydrochloric acid solution, lets cool after adding hot reflux, after filtration, gets subsequent filtrate as need testing solution;

(2) preparation of reference substance solution: get the main active reference substance in appropriate ginkgo leaf medicinal material, comprise rutin, Kaempferol rutinoside, Isorhamnetin rutinoside, Kaempferol, Quercetin, Isorhamnetin, dissolve with water or methyl alcohol or ethanol, be settled to suitable concn, in contrast product solution;

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is acetonitrile, Mobile phase B is 0.1% ~ 2% phosphoric acid solution, gradient elution, flow velocity is 0.9 ~ 1.0ml/min, determined wavelength is 254 ~ 360nm, column temperature is 30-40 DEG C, and writing time is 80-90min;

(4) formulation of standard finger-print: using said method as formulating the means of testing being characterized as main finger-print with ginkgo biloba p.e flavones ingredient; According to the collection of illustrative plates measured by more than 10 batches or 10 batches test samples, formulate standard finger-print, with the retention time of the object of reference chromatographic peak determined for benchmark, calculate the relative retention time of other total chromatographic peak, in described standard finger-print, total peak has 15 ~ 30;

(5) in ginkgo-dipyridamine injection to be measured, be characterized as the means of testing of main finger-print using the described method in (1) ~ (3) with ginkgo biloba p.e flavones ingredient, prepare the finger-print of testing sample;

(6) finger-print of ginkgo-dipyridamine injection to be measured and above-mentioned standard finger-print are contrasted, what should meet the following requirements is part or all of:

I. calculate the finger-print of ginkgo-dipyridamine injection to be measured and the similarity of standard finger-print, should be 0.90 ~ 1.00;

II., in ginkgo-dipyridamine injection finger-print to be measured, non-shared peak area must not exceed 10% of total peak area;

III. the odds ratio of each total peak area in the ratio of the total peak area of 5% ~ 50% and standard finger-print is had comparatively in ginkgo-dipyridamine injection finger-print to be measured, its difference must not be greater than ± and 50%;

B, the test of employing high performance liquid chromatography are based on the finger-print of ginkgo biloba p.e terpene lactone composition characteristics:

(1) preparation of need testing solution: get ginkgo-dipyridamine injection to be measured appropriate, freeze-dried powder is dissolved in water, parenteral solution directly adds 1% ~ 4% hydrochloric acid solution, be extracted with ethyl acetate 3-4 time, divide and get acetic acid ethyl fluid, with 1% ~ 10% sodium acetate solution washing, divide and get sodium acetate liquid, wash with ethyl acetate again, divide and get ethyl acetate washings, wash with water 1-2 time, point water intaking washing lotion, wash with ethyl acetate, combined ethyl acetate liquid, reclaim ethyl acetate to dry, residue adds methyl alcohol makes dissolving, shake up, as need testing solution;

(2) preparation of reference substance solution: get ginkalide A, B, C and Bilobalide in right amount, dissolve with methyl alcohol or ethanol, are settled to suitable concn, in contrast product solution;

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is methyl alcohol, Mobile phase B is tetrahydrofuran: the solution of water=5-15:80-70, gradient elution, flow velocity is 0.7 ~ 0.9ml/min, evaporative light-scattering detector detects, column temperature is within the scope of 25 ~ 35 DEG C, and writing time is 60-70min;

(4) formulation of standard finger-print: using said method as formulating the means of testing being characterized as main finger-print with ginkgo biloba p.e terpene lactone constituents; According to the collection of illustrative plates measured by more than 10 batches or 10 batches test samples, formulate standard finger-print, with the retention time of the object of reference chromatographic peak determined for benchmark, calculate the relative retention time of other total chromatographic peak, in described standard finger-print, total peak has 3 ~ 20;

(5) in ginkgo-dipyridamine injection to be measured, be characterized as the means of testing of main finger-print using the described method in (1) ~ (3) with ginkgo biloba p.e terpene lactone constituents, prepare the finger-print of testing sample;

III. the odds ratio of each total peak area in the ratio of the total peak area of 30% ~ 80% and standard finger-print is had comparatively in ginkgo-dipyridamine injection finger-print to be measured, its difference must not be greater than ± and 50%.

Be preferably:

(1) preparation of need testing solution: it is appropriate that precision takes ginkgo-dipyridamine injection to be measured, wherein parenteral solution is 10ml, freeze-dried powder is the amount containing Dipyridamole 4mg, parenteral solution volatilizes afterwards or freeze-dried powder directly puts methyl alcohol-25% hydrochloric acid solution (4:1) mixed solution 25ml, adds hot reflux 30 minutes, lets cool, filtration is transferred in 25ml measuring bottle, add methyl alcohol to scale mark, shake up, as need testing solution;

(2) preparation of reference substance solution: precision takes rutin, Kaempferol rutinoside, Isorhamnetin rutinoside, Kaempferol, Quercetin, Isorhamnetin, add methyl alcohol and dissolve the solution made every 1ml respectively and contain 0.03mg rutin, 0.02mg Kaempferol rutinoside, 0.02mg Isorhamnetin rutinoside, 0.03mg Kaempferol, 0.03mg Quercetin, 0.02mg Isorhamnetin, product solution in contrast;

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is acetonitrile, Mobile phase B is 0.4% phosphoric acid solution, according to the form below regulation carries out gradient elution, flow velocity is 1.0ml/min, determined wavelength is 360nm, column temperature is 35 DEG C, writing time is 90min, and number of theoretical plate calculates should be not less than 2500 by Quercetin peak;

Table 1 ginkgo-dipyridamine injection flavones ingredient gradient table

(4) formulation of standard finger-print: using said method as formulating the means of testing being characterized as main finger-print with ginkgo biloba p.e flavones ingredient; According to the collection of illustrative plates measured by more than 10 batches or 10 batches test samples, formulate standard finger-print, with the retention time of the object of reference chromatographic peak determined for benchmark, calculate the relative retention time of other total chromatographic peak, in described standard finger-print, total peak has 22;

(6) finger-print of ginkgo-dipyridamine injection to be measured and above-mentioned standard finger-print are contrasted, should meet the following requirements:

I. calculate the finger-print of ginkgo-dipyridamine injection to be measured and the similarity of standard finger-print, should be 0.95 ~ 1.00;

II., in ginkgo-dipyridamine injection finger-print to be measured, non-shared peak area must not exceed 5% of total peak area;

III. the odds ratio of each total peak area in the ratio of the total peak area of 5% ~ 50% and standard finger-print is had comparatively in ginkgo-dipyridamine injection finger-print to be measured, its difference must not be greater than ± and 30%;

(1) preparation of need testing solution: get ginkgo-dipyridamine injection to be measured appropriate, freeze-dried powder adds the water-soluble solution of 20ml containing the amount of Dipyridamole 0.8mg, parenteral solution 20ml directly adds 2% hydrochloric acid solution 2, 4 times are extracted with ethyl acetate jolting, the consumption of each ethyl acetate is respectively 15, 10, 10, 10ml, divide and get acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divide and get sodium acetate liquid, again with ethyl acetate 10ml washing, divide and get ethyl acetate washings, wash 2 times with water, each 20ml, divide water intaking washing lotion, wash with ethyl acetate 10ml, combined ethyl acetate liquid, reclaim ethyl acetate to dry, residue adds methyl alcohol to be made dissolving and is transferred in 5ml measuring bottle, add methyl alcohol to scale, shake up, as need testing solution,

(2) preparation of reference substance solution: get ginkalide A, B, C and Bilobalide are in right amount, accurately weighed, add methyl alcohol and dissolve the solution made every 1ml respectively and contain 1mg ginkalide A, 1mg ginkolide B, 1mg ginkalide C, 2mg Bilobalide, product solution in contrast;

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is methyl alcohol, Mobile phase B is tetrahydrofuran: the solution of water=10:75, according to the form below regulation carries out gradient elution, flow velocity is 0.8ml/min, detect by evaporative light-scattering detector, column temperature is at 30 DEG C, and writing time is 70min; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak;

Table 2 ginkgo-dipyridamine injection terpene lactone composition gradient table

(4) formulation of standard finger-print: using said method as formulating the means of testing being characterized as main finger-print with ginkgo biloba p.e terpene lactone constituents; According to the collection of illustrative plates measured by more than 10 batches or 10 batches test samples, formulate standard finger-print, with the retention time of the object of reference chromatographic peak determined for benchmark, calculate the relative retention time of other total chromatographic peak, in described standard finger-print, total peak has 7;

III. the odds ratio of each total peak area in the ratio of the total peak area of 30% ~ 80% and standard finger-print is had comparatively in ginkgo-dipyridamine injection finger-print to be measured, its difference must not be greater than ± and 30%.

Wherein, content assaying method comprises following content:

The content of flavonoids effective constituent in a, employing high effective liquid chromatography for measuring ginkgo-dipyridamine injection

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is acetonitrile, Mobile phase B is 0.1% ~ 2% phosphoric acid solution, gradient elution, flow velocity is 0.9 ~ 1.1ml/min, determined wavelength is 254 ~ 360nm, column temperature is 30-40 DEG C, and writing time is 80-90min;

(4) determination method: accurate absorption reference substance solution and need testing solution respectively, injection liquid chromatography, measures;

(5) testing result: in the every 10ml Ginkgo Damo injection of this product or every bottle of ginkgo-dipyridamole for injection, quercetin content must not lower than 0.099mg, and Kaempferol content must not lower than 0.166mg, and Isorhamnetin content must not lower than 0.044mg.

The content of terpene lactone effective constituent in b, employing high effective liquid chromatography for measuring ginkgo-dipyridamine injection

(5) testing result: in the every 10ml Ginkgo Damo injection of this product or every bottle of ginkgo-dipyridamole for injection, ginkalide A content must not lower than 0.057mg, ginkolide B content must not lower than 0.021mg, ginkalide C content must not lower than 0.037mg, and content of bilobalide must not lower than 0.081mg.

Be preferably:

(1) preparation of need testing solution: it is appropriate that precision takes ginkgo-dipyridamine injection to be measured, parenteral solution 10ml volatilizes afterwards or freeze-dried powder directly puts methyl alcohol-25% hydrochloric acid solution (4:1) mixed solution 25ml containing the amount of Dipyridamole 4mg, add hot reflux 30 minutes, let cool, filtration is transferred in 25ml measuring bottle, add methyl alcohol to scale mark, shake up, as need testing solution;

Table 3 ginkgo-dipyridamine injection flavones ingredient gradient table

(4) determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures;

(5) testing result: in the every 10ml Ginkgo Damo injection of this product or every bottle of ginkgo-dipyridamole for injection, quercetin content must not lower than 0.105mg, and Kaempferol content must not lower than 0.177mg, and Isorhamnetin content must not lower than 0.048mg.

Table 4 ginkgo-dipyridamine injection terpene lactone composition gradient table

(4) determination method: accurate absorption reference substance solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measures;

(5) testing result: in the every 10ml Ginkgo Damo injection of this product or every bottle of ginkgo-dipyridamole for injection, ginkalide A content must not lower than 0.061mg, ginkolide B content must not lower than 0.022mg, ginkalide C content must not lower than 0.040mg, and content of bilobalide must not lower than 0.087mg.

Compared with prior art, the control ginkgo-dipyridamine injection product quality that the present invention can be more perfect.Because the effective constituent of ginkgo biloba p.e in this product is complicated, if only with wherein one, two kind of composition illustrate its inherent quality, there is certain one-sidedness, more cannot judge the index components of its drug effect, therefore, on the qualitative and quantitative analysis basis that specificity is stronger, increase that to control the quality of ejection preparation based on the finger-print of ginkgo biloba p.e composition characteristics and assay thereof more effective comprehensively; Owing to having ginkgo biloba p.e and Dipyridamole two kinds of compositions in ginkgo-dipyridamine injection, its concrete effective constituent has certain interference each other, adopt conventional liquid phase chromatogram condition, the separating effect at each composition characteristics peak is difficult to meet the demands, only have and adopt condition of the present invention, select suitable parameter to carry out gradient elution, just can obtain exclusive, accurate, stable, workable desirable detection method, more the content limit of concrete effective constituent be controlled.Prove by experiment, the quality control of detection method to the ejection preparation product made with ginkgo biloba p.e, Dipyridamole is more effective, and method precision, stability are all higher.

Experimental example 1: the research being characterized as main finger-print with ginkgo biloba p.e flavones ingredient

A, experimental apparatus, reagent and sample:

Rutin (0080-9705), Kaempferol rutinoside (17650-84-9), Isorhamnetin rutinoside (604-80-8), Kaempferol (110861-200808), Quercetin (10081-200406), Isorhamnetin (110860-200608) reference substance are purchased from Guiyang medicine prison institute; Acetonitrile, methyl alcohol and tetrahydrofuran are chromatographically pure, and it is pure that all the other reagent are analysis; Test Ginkgo Damo injection is provided by Guizhou benefit one hundred company limited.

B, chromatographic condition and system flexibility are tested:

1. the selection of mobile phase:

(1) methyl alcohol-0.1%-2% phosphoric acid water has been investigated respectively, (2) acetonitrile--0.1%-2% phosphoric acid water, (3) acetonitrile in research process--0.1%-2% acetic acid water three kinds of flow phase system, compare through gradient or isocratic elution experiment.Under result display mobile phase (1) condition, peak shape is better, and go out peak completely and be evenly distributed, 6 the composition chromatographic peaks detected can reach baseline separation, shorten analysis time again, good separating effect; Under mobile phase (2) condition, peak shape is better, but appearance time is longer, goes out peak less in 1 hour, goes out peak after many components are trapped in, and is separated not exclusively; Under mobile phase (3) condition, peak shape is poor, is separated bad; So mobile phase (1) is finally selected, wherein methyl alcohol-0.4% phosphoric acid water best results.

2. the selection of chromatographic column:

In research process, conventional octadecylsilane chemically bonded silica has been selected to be the liquid-phase chromatographic column of filling agent, try out SHIMADZU post respectively, DIKMA post, the chromatographic column of Agela post and Agilent post four kinds of trades mark, the chromatographic column peak shape that result shows four kinds of trades mark has different, SHIMADZU post and DIKMA post, Agela post, Agilent post differ greatly, the peak shape of Agela post is better, go out peak retention time to differ greatly, separating effect Agela post is better than HIMADZU post and DIKMA post, Agilent post, therefore determines that Agela post is as fingerprint map analyzing post.

3. the investigation of column temperature

Chromatographic condition is determined according to above-mentioned, respectively column temperature is set to 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C to investigate, when column temperature often raises 5 DEG C, appearance time reach 2min, peak integral translation, but peak shape is constant, when only having 40 DEG C, peak shape has and necessarily moves down, and illustrates that durability is good, does not have an impact to analysis, under 35 DEG C of column temperatures, baseline is comparatively steady, therefore selects 35 DEG C of column temperatures.

4. the investigation of flow velocity

Chromatographic condition is determined according to above-mentioned, flow velocity is set to for 0.9ml/min, 1.0ml/min, 1.1ml/min investigate respectively, the change of flow velocity only have impact on appearance time, but peak shape is basically identical, illustrate that the durability of chromatographic condition is good, 0.9ml/min chromatogram baseline has and necessarily moves down, and 1.1ml/min flow velocity is comparatively large to pillar pressure, therefore finally determines that flow velocity is 1.0ml/min.

5. the determination of determined wavelength:

In research under methyl alcohol-0.4% phosphoric acid water mobile phase condition, investigated respectively different-waveband typical wavelengths 215,254,280, chromatographic peak situation under 360nm, result shows, under 215nm, peak shape and baseline are not steady, with 254,280, appearance time peak shape under 360nm is inconsistent, 254, under 280nm, 360nm, baseline is good, appearance time is basically identical, but 254, under 280nm, peak shape is less, be not easy to observation analysis, 360nm is mainly the maximum absorption wavelength of flavones, therefore final choice 360nm is as determined wavelength.

6. the selection of writing time

Prepare need testing solution by selected method, draw 10 μ L, injection liquid chromatography, by gradient condition, measure in accordance with the law, record 120min chromatogram.Chromatogram occurs without cutting edge of a knife or a sword shape after 75min, and go out after peak figure compares with the solvent of independent sample introduction, solvent goes out peak to it not to be affected, therefore determines record 80min chromatogram.

7. determine chromatographic condition:

Mobile phase: acetonitrile: 0.4% phosphoric acid water (gradient), chromatographic column is Agela post, and column temperature is 35 DEG C, and wavelength is 360nm, and flow velocity is 1.0ml/min.

Eluent gradient:

Table 5 ginkgo-dipyridamine injection flavones ingredient gradient table

8. the preparation of need testing solution:

Get Ginkgo Damo injection 10ml, volatilize rearmounted methyl alcohol-25% hydrochloric acid solution (4:1) mixed solution 25ml, add hot reflux 30 minutes, let cool, filter and be transferred in 25ml measuring bottle, add methyl alcohol to scale mark, shake up, to obtain final product.

9. the preparation of object of reference solution:

Quercetin is one of ginkgo biloba p.e main active, its integral area proportion in finger-print more greatly and relatively stable, takes into account the research of intermediate and bulk drug simultaneously, and therefore selected Quercetin is as object of reference, and with methanol as solvent, it is feasible that measurement result shows the method.

10. finger-print stability, precision, repeated experiment

10.1 stability: get need testing solution, sample 10 μ L (0h, 2h, 4h, 8h, 12h) in different time, injection liquid chromatography, measures in accordance with the law.Result shows, each main chromatographic peak relative retention time and peak area account for total peak area 5% and have no significant change (determining 19 common peaks) with the peak area ratio of superiors, its RSD is respectively 0.01%-0.22% and 0.26%-2.27%, meet fingerprint pattern technology requirement (RSD is all within 3%), have good stability.And after " similarity evaluation A version " calculates, its similarity reaches more than 0.99.

10.2 precision: get need testing solution, accurate absorption 10 μ L, injection liquid chromatography, repeats sample introduction 6 times, record finger-print.Result shows, each main chromatographic peak relative retention time and peak area account for total peak area 5% and have no significant change (determining 19 common peaks) with the peak area ratio of superiors, its RSD is respectively 0.01%-0.19% and 0.33%-2.11%, meet fingerprint pattern technology requirement (RSD is all within 3%), precision is good.And after " similarity evaluation A version " calculates, its similarity reaches more than 0.99.

10.3 repeatability: the test sample 6 parts getting same lot number, be prepared as stated above and detect, investigate the consistance of each chromatographic peak relative time, peak area ratio, result shows, each main chromatographic peak relative retention time has no significant change (determining 19 common peaks) with the relative peak area accounting for total peak area more than 5% chromatographic peak, its RSD is respectively 0.02%-0.36% and 0.60%-2.59%, meets fingerprint pattern technology requirement (RSD is all within 3%), and repeatability is good.And after " similarity evaluation A version " calculates, its similarity reaches more than 0.99.

The formulation of 11. finger-prints and technical parameter:

11.1 reference fingerprints: according to 10 batches of Ginkgo Damo injection sample finger-prints, with Quercetin reference substance for object of reference, formulate standard finger-print, compared with standard finger-print by test sample finger-print, similarity is all between 0.95 ~ 1.00.

The demarcation of 11.2 total fingerprint peakses

According to the testing result of 10 batches of Ginkgo Damo injection finger-prints, see Fig. 1.Have 22 peaks all can better be separated, be numbered respectively, wherein 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, S, 16,17,18,19,20, No. 21 peaks all occur at identical relative retention time place in 10 batches of Ginkgo Damo injections, its finger-print area sum accounts for total amount and is all greater than 95%, thus demarcates these 22 chromatographic peaks for total fingerprint peaks.Wherein pointed out 7 chromatographic peaks by reference substance Comparability test, peak 5,10,11, S, 17,18,20 are followed successively by rutin respectively, Kaempferol rutinoside, Isorhamnetin rutinoside, and Quercetin reaches not, Kaempferol, Isorhamnetin.

The peak area ratio of 11.3 total fingerprint peakses

5,10, No. 11 peak (rutins, Kaempferol rutinoside, Isorhamnetin rutinoside) account for total peak area be greater than the 5% total peak being less than 10% for first three ten minutes unit goes out peak area, its difference is respectively+11%-14% ,+13%-22%, + 13%-16%, meets " technical requirement of traditional Chinese medicine finger-print research " (provisional) and must not be greater than positive and negative 30%.S, No. 18 peaks (Quercetin, Kaempferol) account for for latter 30 minutes units go out peak area the total peak that total peak area is greater than 10%, and its difference is respectively+12%-14%, + 9%-12%, meets " technical requirement of traditional Chinese medicine finger-print research " (provisional) and must not be greater than positive and negative 25%.All the other first three ten minutes total peak unit peak areas account for total peak area all lower than 5%, within latter 30 minutes, total peak unit peak area accounts for total peak area all lower than 10%, according to " technical requirement of traditional Chinese medicine finger-print research " (provisional), its peak area ratio does not do requirement.

11.4 non-shared peak area

10 batches of Ginkgo Damo injections HPLC spectrogram measurement result separately shows, non-shared peak area accounts for total peak area and is less than 5%, meets " technical requirement of traditional Chinese medicine finger-print research " (provisional).

Experimental example 2 is based on the research of the finger-print of ginkgo biloba p.e terpene lactone composition characteristics

A, experimental apparatus, reagent and sample:

Ginkalide A (110862-200305), ginkolide B (110863-200305), ginkalide C (110864-200), Bilobalide (110865-200404) reference substance are purchased from Guiyang medicine prison institute; Acetonitrile, methyl alcohol and tetrahydrofuran are chromatographically pure, and it is pure that all the other reagent are analysis; Test Ginkgo Damo injection is provided by Guizhou benefit one hundred company limited.

B, chromatographic condition and system flexibility are tested:

1. the selection of mobile phase:

(1) methyl alcohol has been investigated respectively: (tetrahydrofuran: water) in research process, (2) acetonitrile: (tetrahydrofuran: water), (3) isopropyl alcohol: methyl alcohol: water three kinds of flow phase system, compares through gradient or isocratic elution experiment.Under result display mobile phase (1) condition, peak shape is better, and go out peak completely and be evenly distributed, 4 the composition chromatographic peaks detected can reach baseline separation, shorten analysis time again, good separating effect; Under mobile phase (2) condition, peak shape is better, but appearance time is longer; Under mobile phase (3) condition, peak shape is poor, is separated bad; So mobile phase (1) is finally selected, wherein tetrahydrofuran: water=10:75 best results.

2. the selection of chromatographic column:

In research process, use Kromasil post respectively, DIKMA post, Agela post, Agilent post is analyzed, and compares separating resulting, and four kinds of chromatographic columns are compared, peak shape has different, DIKMA post and Kromasil post, Agela post, Agilent post differ greatly, and the peak shape of Agela post is better, goes out peak retention time and differs greatly, separating effect Agela post is better than HIMADZU post and DIKMA post, Agilent post, therefore determines that Agela post is as fingerprint map analyzing post.

3. the investigation of column temperature

Chromatographic condition is determined according to above-mentioned, respectively column temperature is set to 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C to investigate, when column temperature often raises 5 DEG C, appearance time reach 1min, peak integral translation, but peak shape is constant, 25 DEG C of column temperature base lines are steady, and necessarily move down when column temperature has to peak shape when 35 DEG C and 40 DEG C, general description durability is good, analysis is not had an impact, therefore selects 30 DEG C of column temperatures.

4. the investigation of flow velocity

Flow velocity is set to for 0.7ml/min, 0.8ml/min, 0.9ml/min investigate respectively in research process, the change of flow velocity only have impact on appearance time, but peak shape is basically identical, illustrate that the durability of chromatographic condition is good, comparatively 0.8ml/min is not steady for the chromatogram baseline of 0.7ml/min and 0.9ml/min flow velocity, therefore finally determines that flow velocity is 0.8ml/min.

5. the selection of writing time

Prepare need testing solution by selected method, draw 20 μ L, injection liquid chromatography, by gradient condition, measure in accordance with the law, record 60min chromatogram.Chromatogram occurs without cutting edge of a knife or a sword shape after 50min, and go out after peak figure compares with the solvent of independent sample introduction, solvent goes out peak to it not to be affected, therefore determines record 60min chromatogram.

6. determine chromatographic condition:

Mobile phase: methyl alcohol: (tetrahydrofuran: water) (10:75) (gradient), chromatographic column is Agela post, and column temperature is 30 DEG C, and flow velocity is 0.8ml/min.Evaporative light condition is temperature: 50 DEG C, pressure: 3.5bar, gain: 7, wave filter: 5s, light source: open.

Eluent gradient:

Table 6 ginkgo-dipyridamine injection terpene lactone composition gradient table

7. the preparation of need testing solution:

Get Ginkgo Damo injection 20ml, precision measures, and adds 2% hydrochloric acid solution 2, with ethyl acetate jolting extract 4 times (15,10,10,10ml), point get acetic acid ethyl fluid, wash with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, then wash with ethyl acetate 10ml.Divide and get ethyl acetate washing lotion, wash 2 times with water, each 20ml, point water intaking washing lotion, washs with ethyl acetate 10ml, merges ethyl acetate liquid, and recovery of acetic acid ethyl ester is to doing, and residue methyl alcohol dissolves and is transferred in 5ml measuring bottle, adds methyl alcohol to scale, shakes up, to obtain final product.

8. the preparation of object of reference solution:

Bilobalide is one of ginkgo biloba p.e lactone active component, proportion is more greatly and relatively stable in finger-print for its integral area, take into account the research of intermediate and bulk drug simultaneously, therefore selected Bilobalide is as object of reference, and with methanol as solvent, it is feasible that measurement result shows the method.

9. finger-print stability, precision, repeated experiment

9.1 stability: get need testing solution, sample 20 μ L (0h, 1h, 4h, 8h, 12h) in different time, injection liquid chromatography, measures in accordance with the law.Result shows, each main chromatographic peak relative retention time and peak area account for total peak area 5% and have no significant change (determining 7 common peaks) with the peak area ratio of superiors, its RSD is respectively 0.23%-0.44% and 0.43%-1.83%, meet fingerprint pattern technology requirement (RSD is all within 3%), have good stability.And after " similarity evaluation A version " calculates, its similarity reaches more than 0.99.

9.2 precision: get need testing solution, accurate absorption 20 μ L, injection liquid chromatography, repeats sample introduction 6 times, record finger-print.Result shows, each main chromatographic peak relative retention time and peak area account for total peak area 5% and have no significant change (determining 7 common peaks) with the peak area ratio of superiors, its RSD is respectively 0.18%-0.40% and 0.28%-2.08%, meet fingerprint pattern technology requirement (RSD is all within 3%), precision is good.And after " similarity evaluation A version " calculates, its similarity reaches more than 0.99.

9.3 repeatability: the test sample 6 parts getting same lot number, be prepared as stated above and detect, investigate the consistance of each chromatographic peak relative time, peak area ratio, result shows, each main chromatographic peak relative retention time has no significant change (determining 7 common peaks) with the relative peak area accounting for total peak area more than 5% chromatographic peak, its RSD is respectively 0.04%-0.29% and 0.50%-2.48%, meets fingerprint pattern technology requirement (RSD is all within 3%), and repeatability is good.And after " similarity evaluation A version " calculates, its similarity reaches more than 0.99.

10. the formulation of finger-print and technical parameter:

10.1 reference fingerprints: according to 10 batches of Ginkgo Damo injection sample finger-prints, with Bilobalide reference substance for object of reference, formulate standard finger-print, compared with standard finger-print by test sample finger-print, similarity is all between 0.95 ~ 1.00.

The demarcation of 10.2 total fingerprint peakses

According to the testing result of 10 batches of Ginkgo Damo injection finger-prints, see Fig. 2.Have 7 peaks all can better be separated, be numbered respectively, wherein 1,2, S, 3,4,5, No. 6 peaks all occur at identical relative retention time place in 10 batches of Ginkgo Damo injections, its finger-print area sum accounts for total amount and is all greater than 95%, thus demarcates these 7 chromatographic peaks for total fingerprint peaks.Wherein pointed out 4 chromatographic peaks by reference substance Comparability test, peak 2, S, 3,4 are followed successively by ginkalide C, Bilobalide, ginkalide A, ginkolide B respectively.

The peak area ratio of 10.3 total fingerprint peakses

S peak (Bilobalide) accounts for for first three ten minutes unit goes out peak area the total peak that total peak area is greater than 20%, and its difference is+10%-9%, meets " technical requirement of traditional Chinese medicine finger-print research " (provisional) and must not be greater than positive and negative 20%.No. 1, No. 2 peaks (ginkalide C) account for total peak area be more than or equal to the 5% total peak being less than 10% for first three ten minutes unit go out peak area, its difference is respectively+17%-12% ,+17%-14%, meets " technical requirement of traditional Chinese medicine finger-print research " (provisional) and must not be greater than positive and negative 30%.No. 3 peaks (ginkalide A) account for total peak area be more than or equal to the 10% total peak being less than 20% for first three ten minutes unit go out peak area, its difference is+12%-15%, meets " technical requirement of traditional Chinese medicine finger-print research " (provisional) and must not be greater than positive and negative 25%.No. 6 peaks be after 30 minutes units go out peak area and account for the total peak that total peak area is greater than 10%, its difference be+9%-8%, meets " technical requirement that traditional Chinese medicine finger-print is studied " (provisional) and must not be greater than positive and negative 25%.All the other first three ten minutes total peak unit peak areas account for total peak area all lower than 5%, within latter 30 minutes, total peak unit peak area accounts for total peak area all lower than 10%, according to " technical requirement of traditional Chinese medicine finger-print research " (provisional), its peak area ratio does not do requirement.

10.4 non-shared peak area

Experimental example 3 ginkgo-dipyridamine injection assay is studied

1. instrument and reagent

1.1 instruments: analytical balance (plum Teller), nutsch filter, high performance liquid chromatograph (Agilent1100 type, band diode array detector, evaporative light detecting device).

1.2 reagents: Kaempferol (110861-200808), Quercetin (10081-200406), Isorhamnetin (110860-200608), ginkalide A (110862-200305), ginkolide B (110863-200305), ginkalide C (110864-200), Bilobalide (110865-200404) reference substance are purchased from Guiyang medicine prison institute; Acetonitrile, methyl alcohol and tetrahydrofuran are chromatographically pure, and it is pure that all the other reagent are analysis; Test Ginkgo Damo injection is provided by Guizhou benefit one hundred company limited.

2. flavonoids multi-target ingredient assay research

2.1 chromatographic condition

Measure with Ginkgo Damo injection finger-print flavonoids.

The preparation of 2.2 need testing solutions

2.3 linear relationship

Precision takes Quercetin, Kaempferol, Isorhamnetin are appropriate, adds methyl alcohol and dissolves and make mass concentration and be respectively 0.137mg/ml, the reference substance solution of 0.090mg/ml, 0.067mg/ml, inject HPLC measure by the chromatographic condition drafted.Quercetin sample size is respectively 2,6,10,14,18 μ l, and Kaempferol sample size is respectively 15,20,25,30,35 μ l, and Isorhamnetin sample size is respectively 5,10,15,20,25 μ l.With peak area, linear regression is carried out to sample introduction quality, obtain the regression equation of each tested composition, related coefficient and the range of linearity.

Table 7 flavonoids effective constituent linear relationship table

2.4 assay stability, precision, repeatability, the recovery are tested

Stability: prepare preparation need testing solution according to Ginkgo Damo injection need testing solution preparation method respectively, by 0h, 2h, 4h, 8h, 12h sample introduction 10 μ l respectively, measures each index components peak area.Result shows in need testing solution 12h stable.Its RSD is respectively 1.15%, 0.83% and 0.97%, meets quality standard technical requirement (RSD is all within 3%), has good stability.

Precision: get preparation need testing solution 10 μ l respectively, continuous sample introduction 6 times, measure peak area, 6 peak area RSD are between 0.62%-0.76% as a result, and meet quality standard technical requirement (RSD is all within 3%), precision is good.

Repeatability: the Ginkgo Damo injection 6 parts getting same lot number, prepares need testing solution 10 μ l according to need testing solution preparation method, measures respectively, calculate each index content, RSD, between 1.10%-1.35%, meets quality standard technical requirement (RSD is all within 3%), and repeatability is good.

The recovery: adopt average recovery method, by above-mentioned sample solution preparation method, sample is processed, add appropriate Quercetin, Kaempferol, Isorhamnetin reference substance solution (0.161mg/ml, 0.401mg/ml, 0.155mg/ml), make basic, normal, high 3 kinds of variable concentrations need testing solutions, measure respectively, average recovery rate is 100.13%, RSD is 2.57%, meets quality standard technical requirement (RSD is all within 3%).

3. flavonoids multi-target ingredient assay research

3.1 chromatographic condition

Measure with Ginkgo Damo injection finger-print lactone.

The preparation of 3.2 need testing solutions

Get Ginkgo Damo injection and be about 20ml, precision measures, and adds 2% hydrochloric acid solution 2, with ethyl acetate jolting extract 4 times (15,10,10,10ml), point get acetic acid ethyl fluid, wash with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, then wash with ethyl acetate 10ml.Divide and get ethyl acetate washing lotion, wash 2 times with water, each 20ml, point water intaking washing lotion, washs with ethyl acetate 10mL, merges ethyl acetate liquid, and recovery of acetic acid ethyl ester is to doing, and residue methyl alcohol dissolves and is transferred in 5ml measuring bottle, adds methyl alcohol to scale, shakes up, to obtain final product.

3.3 linear relationship

It is appropriate that precision takes Ginkgolide A. B. C and Bilobalide, adds methyl alcohol and dissolve and make mass concentration and be respectively 0.425mg/ml, the reference substance solution of 0.103mg/ml, 0.097mg/ml, 0.163mg/ml, inject HPLC measure by the chromatographic condition drafted.Ginkalide A sample size is respectively 1,3,5,7,9 μ l, ginkolide B sample size is respectively 2,4,6,8,10 μ l, ginkalide C sample size is respectively 4,8,12,16,20 μ l, and Bilobalide sample size is respectively, 5,10,15,20,25 μ l.With peak area, linear regression is carried out to sample introduction quality, obtain the regression equation of each tested composition, related coefficient and the range of linearity.

Table 8 terpene lactone effective constituent linear relationship table

3.4 assay stability, precision, repeatability, the recovery are tested

Stability: prepare preparation need testing solution according to Ginkgo Damo injection need testing solution preparation method respectively, by 0h, 1h, 4h, 8h, 12h sample introduction 20 μ l respectively, measures each index components peak area.Result shows in need testing solution 12h stable.Its RSD is respectively 0.56%, 0.82% and 1.83%, meets quality standard technical requirement (RSD is all within 3%), has good stability.

Precision: get preparation need testing solution 20 μ l respectively, continuous sample introduction 6 times, measure peak area, 6 peak area RSD are between 0.62%-2.08% as a result, and meet quality standard technical requirement (RSD is all within 3%), precision is good.

Repeatability: the Ginkgo Damo injection 6 parts getting same lot number, prepares need testing solution 20 μ l according to need testing solution preparation method, measures respectively, calculate each index content, RSD, between 0.49%-2.48%, meets quality standard technical requirement (RSD is all within 3%), and repeatability is good.

The recovery: adopt average recovery method, by above-mentioned sample solution preparation method, sample is processed, add appropriate Ginkgolide A. B. C and Bilobalide reference substance solution (0.0198mg/ml, 0.0109mg/ml, 0.0201mg/ml, 0.0409mg/ml), make basic, normal, high 3 kinds of variable concentrations need testing solutions, measure respectively, average recovery rate is 100.11%, RSD is 2.85%, meets quality standard technical requirement (RSD is all within 3%).

4. assay result

Get the Ginkgo Damo injection of 10 batches respectively, the chromatographic condition drafted by need testing solution preparation method, measure the content of 3 principal ingredients, measurement result is in Table.

Table 9 Ginkgo Damo injection assay result (mg/ bottle (10ml))

Result shows, our company's Quercetin average out to 0.124mg/ bottle in 10 batches of Ginkgo Damo injection samples of production in 2009 to 2012; Kaempferol average out to 0.208mg/ bottle; Isorhamnetin average out to 0.055mg/ bottle; Ginkalide A average out to 0.072mg/ bottle; Ginkolide B average out to 0.026mg/ bottle; Ginkalide C average out to 0.047mg/ bottle; Bilobalide average out to 0.102mg/ bottle.Comprehensive analysis 10 batches of Ginkgo Damo injection finished product measurement results, because each batch of ginkgo leaf crude drug has different, and then have influence on the content of each effective constituent in Ginkgo Damo injection, therefore each composition measurement standard is decided to be Quercetin is not less than 0.099mg/ bottle, Kaempferol is not less than 0.166mg/ bottle, Isorhamnetin is not less than 0.044mg/ bottle, ginkalide A is not less than 0.057mg/ bottle, ginkolide B is not less than 0.021mg/ bottle, ginkalide C is not less than 0.037mg/ bottle, and Bilobalide is not less than 0.081mg/ bottle.

Accompanying drawing explanation

Fig. 1 is Ginkgo Damo injection flavonoids finger-print of the present invention research chromatogram;

Fig. 2 is Ginkgo Damo injection terpene lactone finger-print of the present invention research chromatogram.

Embodiment

Below in conjunction with embodiment, technical scheme of the present invention is further described.

Embodiment 1: adopt in liquid chromatography for measuring Ginkgo Damo injection and be characterized as main finger-print with ginkgo biloba p.e flavones ingredient:

(1) preparation of need testing solution: precision takes Ginkgo Damo injection 10ml to be measured, volatilizes rearmounted methyl alcohol-25% hydrochloric acid solution (4:1) mixed solution 25ml, adds hot reflux 30 minutes, let cool, filter and be transferred in 25ml measuring bottle, add methyl alcohol to scale mark, shake up, as need testing solution;

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler; Mobile phase A is acetonitrile, and Mobile phase B is 0.4% phosphoric acid solution, gradient elution, and order is: 0-30min, 85%-83%B; 30-40min, 83%-80%B; 40-60min, 80%-75%B; 60-80min, 75%-20%B; 80-90min, 20%-85%B; Flow velocity is 1.0ml/min, determined wavelength is 360nm, column temperature is 35 DEG C, and writing time is 90min, and number of theoretical plate calculates should be not less than 2500 by Quercetin peak;

(5) in Ginkgo Damo injection to be measured, be characterized as the means of testing of main finger-print using the described method in (1) ~ (3) with ginkgo biloba p.e flavones ingredient, prepare the finger-print of testing sample;

(6) Ginkgo Damo injection finger-print to be measured and above-mentioned standard finger-print are contrasted, meet the following requirements:

I. calculate the finger-print of Ginkgo Damo injection to be measured and the similarity of standard finger-print, should be 0.95 ~ 1.00;

II., in Ginkgo Damo injection finger-print to be measured, non-shared peak area must not exceed 5% of total peak area;

III. the odds ratio of each total peak area in the ratio of the total peak area of 5% ~ 50% and standard finger-print is had comparatively in Ginkgo Damo injection finger-print to be measured, its difference must not be greater than ± and 30%.

Embodiment 2: based on the finger-print of ginkgo biloba p.e terpene lactone composition characteristics in employing liquid chromatography for measuring Ginkgo Damo injection:

(1) preparation of need testing solution: get Ginkgo Damo injection 20ml to be measured, add 2% hydrochloric acid solution 2, 4 times are extracted with ethyl acetate jolting, the consumption of each ethyl acetate is respectively 15, 10, 10, 10ml, divide and get acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divide and get sodium acetate liquid, again with ethyl acetate 10ml washing, divide and get ethyl acetate washings, wash 2 times with water, each 20ml, divide water intaking washing lotion, wash with ethyl acetate 10ml, combined ethyl acetate liquid, reclaim ethyl acetate to dry, residue adds methyl alcohol to be made dissolving and is transferred in 5ml measuring bottle, add methyl alcohol to scale, shake up, as need testing solution,

(3) chromatographic condition: mobile phase A is methyl alcohol, Mobile phase B is tetrahydrofuran: the solution of water=10:75, gradient elution, and order is: 0-20min, 75%-75%B; 20-60min, 75%-32%B; 60-70min, 32%-75%B; Flow velocity is 0.8ml/min, and detect by evaporative light-scattering detector, column temperature is at 30 DEG C, and writing time is 70min; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak;

(5) in Ginkgo Damo injection to be measured, be characterized as the means of testing of main finger-print using the described method in (1) ~ (3) with ginkgo biloba p.e terpene lactone constituents, prepare the finger-print of testing sample;

(6) finger-print of Ginkgo Damo injection to be measured and above-mentioned standard finger-print are contrasted, meet the following requirements:

III. the odds ratio of each total peak area in the ratio of the total peak area of 30% ~ 80% and standard finger-print is had comparatively in Ginkgo Damo injection finger-print to be measured, its difference must not be greater than ± and 30%.

Embodiment 3: adopt in liquid chromatography for measuring ginkgo-dipyridamole for injection and be characterized as main finger-print with ginkgo biloba p.e flavones ingredient:

(1) preparation of need testing solution: it is the amount containing Dipyridamole 4mg that precision takes ginkgo Damo freeze-drying powder to be measured, directly put methyl alcohol-25% hydrochloric acid solution (4:1) mixed solution 25ml, add hot reflux 30 minutes, let cool, filtration is transferred in 25ml measuring bottle, add methyl alcohol to scale mark, shake up, as need testing solution;

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler; Mobile phase A is acetonitrile, and Mobile phase B is 2% phosphoric acid solution, gradient elution, and order is: 0-30min, 85%-83%B; 30-40min, 83%-80%B; 40-60min, 80%-75%B; 60-80min, 75%-20%B; 80-90min, 20%-85%B; Flow velocity is 1.0ml/min, determined wavelength is 240nm, column temperature is 30 DEG C, and writing time is 80min;

(5) in ginkgo-dipyridamole for injection to be measured, be characterized as the means of testing of main finger-print using the described method in (1) ~ (3) with ginkgo biloba p.e flavones ingredient, prepare the finger-print of testing sample;

(6) ginkgo-dipyridamole for injection finger-print to be measured and above-mentioned standard finger-print are contrasted, meet the following requirements:

I. calculating the finger-print of ginkgo-dipyridamole for injection to be measured and the similarity of standard finger-print, is 0.90 ~ 1.00;

II., in ginkgo-dipyridamole for injection finger-print to be measured, non-shared peak area is no more than 10% of total peak area;

Embodiment 4: based on the finger-print of ginkgo biloba p.e terpene lactone composition characteristics in employing liquid chromatography for measuring ginkgo-dipyridamole for injection:

(1) preparation of need testing solution: getting ginkgo Damo freeze-drying powder to be measured is that the amount containing Dipyridamole 0.8mg adds the water-soluble solution of 20ml, add 2% hydrochloric acid solution 2, 4 times are extracted with ethyl acetate jolting, the consumption of each ethyl acetate is respectively 15, 10, 10, 10ml, divide and get acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divide and get sodium acetate liquid, again with ethyl acetate 10ml washing, divide and get ethyl acetate washings, wash 2 times with water, each 20ml, divide water intaking washing lotion, wash with ethyl acetate 10ml, combined ethyl acetate liquid, reclaim ethyl acetate to dry, residue adds methyl alcohol to be made dissolving and is transferred in 5ml measuring bottle, add methyl alcohol to scale, shake up, as need testing solution,

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler; Mobile phase A is methyl alcohol, and Mobile phase B is tetrahydrofuran: the solution of water=15:70, gradient elution, and order is: 0-20min, 75%-75%B; 20-60min, 75%-32%B; 60-70min, 32%-75%B; Flow velocity is 0.9ml/min, and detect by evaporative light-scattering detector, column temperature is at 25 DEG C, and writing time is 70min;

(5) in ginkgo-dipyridamole for injection to be measured, be characterized as the means of testing of main finger-print using the described method in (1) ~ (3) with ginkgo biloba p.e terpene lactone constituents, prepare the finger-print of testing sample;

(6) finger-print of ginkgo-dipyridamole for injection to be measured and above-mentioned standard finger-print are contrasted, meet the following requirements:

I. calculate the finger-print of ginkgo-dipyridamole for injection to be measured and the similarity of standard finger-print, should be 0.90 ~ 1.00;

II. the odds ratio of each total peak area in the ratio of the total peak area of 30% ~ 80% and standard finger-print is had comparatively in ginkgo-dipyridamole for injection finger-print to be measured, its difference must not be greater than ± and 50%.

Embodiment 5: the content adopting flavonoids effective constituent in liquid chromatography for measuring Ginkgo Damo injection:

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is acetonitrile, and Mobile phase B is 0.4% phosphoric acid solution, gradient elution, order is: 0-30min, 85%-83%B; 30-40min, 83%-80%B; 40-60min, 80%-75%B; 60-80min, 75%-20%B; 80-90min, 20%-85%B; Flow velocity is 1.0ml/min, determined wavelength is 360nm, column temperature is 35 DEG C, and writing time is 90min, and number of theoretical plate calculates should be not less than 2500 by Quercetin peak;

(5) testing result: in the every 10ml Ginkgo Damo injection of this product, quercetin content must not lower than 0.099mg, and Kaempferol content must not lower than 0.166mg, and Isorhamnetin content must not lower than 0.044mg.

Embodiment 6: the content adopting terpene lactone effective constituent in liquid chromatography for measuring Ginkgo Damo injection:

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is methyl alcohol, and Mobile phase B is tetrahydrofuran: the solution of water=10:75, gradient elution, order is 0-20min, 75%-75%B; 20-60min, 75%-32%B; 60-70min, 32%-75%B; Flow velocity is 0.8ml/min, and detect by evaporative light-scattering detector, column temperature is at 30 DEG C, and writing time is 70min; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak;

(5) testing result: in the every 10ml Ginkgo Damo injection of this product, ginkalide A content must not lower than 0.057mg, ginkolide B content must not lower than 0.021mg, ginkalide C content must not lower than 0.037mg, and content of bilobalide must not lower than 0.081mg.

Embodiment 7: the content adopting flavonoids effective constituent in liquid chromatography for measuring ginkgo-dipyridamole for injection:

(1) preparation of need testing solution: precision takes the amount of ginkgo Damo freeze-drying powder to be measured containing Dipyridamole 4mg, directly put methyl alcohol-25% hydrochloric acid solution (4:1) mixed solution 25ml, add hot reflux 30 minutes, let cool, filtration is transferred in 25ml measuring bottle, add methyl alcohol to scale mark, shake up, as need testing solution;

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is acetonitrile, and Mobile phase B is 0.1% phosphoric acid solution, gradient elution, order is: 0-30min, 85%-83%B; 30-40min, 83%-80%B; 40-60min, 80%-75%B; 60-80min, 75%-20%B; 80-90min, 20%-85%B; Flow velocity is 1.0ml/min, determined wavelength is 280nm, column temperature is 40 DEG C, and writing time is 90min;

(5) testing result: in this product every bottle ginkgo-dipyridamole for injection (specification: in Dipyridamole 4mg), quercetin content must not lower than 0.105mg, and Kaempferol content must not lower than 0.177mg, and Isorhamnetin content must not lower than 0.048mg.

Embodiment 8: the content adopting terpene lactone effective constituent in liquid chromatography for measuring ginkgo-dipyridamole for injection:

(1) preparation of need testing solution: getting ginkgo Damo freeze-drying powder to be measured is the amount containing Dipyridamole 0.8mg, add the water-soluble solution of 20ml, add 2% hydrochloric acid solution 2, 4 times are extracted with ethyl acetate jolting, the consumption of each ethyl acetate is respectively 15, 10, 10, 10ml, divide and get acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divide and get sodium acetate liquid, again with ethyl acetate 10ml washing, divide and get ethyl acetate washings, wash 2 times with water, each 20ml, divide water intaking washing lotion, wash with ethyl acetate 10ml, combined ethyl acetate liquid, reclaim ethyl acetate to dry, residue adds methyl alcohol to be made dissolving and is transferred in 5ml measuring bottle, add methyl alcohol to scale, shake up, as need testing solution,

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler: mobile phase A is methyl alcohol, and Mobile phase B is tetrahydrofuran: the solution of water=5:80, gradient elution, order is 0-20min, 75%-75%B; 20-60min, 75%-32%B; 60-70min, 32%-75%B; Flow velocity is 0.7ml/min, and detect by evaporative light-scattering detector, column temperature is at 35 DEG C, and writing time is 70min;

(5) testing result: in this product every bottle ginkgo-dipyridamole for injection (specification: in Dipyridamole 4mg), ginkalide A content must not lower than 0.061mg, ginkolide B content must not lower than 0.022mg, ginkalide C content must not lower than 0.040mg, and content of bilobalide must not lower than 0.087mg.

Claims

1. a detection method for ginkgo-dipyridamine injection, the injection that described preparation is prepared from by ginkgo biloba p.e 20-100 weight portion, Dipyridamole 2-10 parts by weight of composition, is characterized in that: the method comprises following content:

2., according to the detection method of ginkgo-dipyridamine injection according to claim 1, it is characterized in that finger-print comprises following content:

(6) finger-print of ginkgo-dipyridamine injection to be measured and above-mentioned standard finger-print are contrasted, part or all of in should meeting the following requirements:

3., according to the detection method of ginkgo-dipyridamine injection according to claim 2, it is characterized in that finger-print comprises following content:

(3) chromatographic condition: chromatographic column adopts octadecylsilane chemically bonded silica to be filler; Mobile phase A is methyl alcohol, and Mobile phase B is tetrahydrofuran: the solution of water=10:75, gradient elution, and order is: 0-20min, 75%-75%B; 20-60min, 75%-32%B; 60-70min, 32%-75%B; Flow velocity is 0.8ml/min, and detect by evaporative light-scattering detector, column temperature is at 30 DEG C, and writing time is 70min; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak;

4. according to the detection method of ginkgo-dipyridamine injection according to claim 1, it is characterized in that: assay comprises following content:

The content of flavonoids effective constituent in a, employing high effective liquid chromatography for measuring ginkgo-dipyridamine injection:

(5) testing result: in the every 10ml Ginkgo Damo injection of this product or every bottle of ginkgo-dipyridamole for injection, quercetin content must not lower than 0.099mg, and Kaempferol content must not lower than 0.166mg, and Isorhamnetin content must not lower than 0.044mg;

The content of terpene lactone effective constituent in b, employing high effective liquid chromatography for measuring ginkgo-dipyridamine injection:

5. according to the detection method of ginkgo-dipyridamine injection according to claim 4, it is characterized in that: assay comprises following content:

(5) testing result: in the every 10ml Ginkgo Damo injection of this product or every bottle of ginkgo-dipyridamole for injection, quercetin content must not lower than 0.105mg, and Kaempferol content must not lower than 0.177mg, and Isorhamnetin content must not lower than 0.048mg;

6., according to the detection method of the arbitrary described ginkgo-dipyridamine injection of claim 1-5, it is characterized in that: described ejection preparation be directly used in drug administration by injection parenteral solution, need dilute after for the concentrated solution for injection of drip-feed, the injection sterile powder directly obtained for the glucose intravenous infusion of drip-feed or sodium chloride intravenous infusion or freeze-drying or spray drying process or aseptic block.