CN104569170B

CN104569170B - A kind of detection method of ginkgo-dipyridamine injection

Info

Publication number: CN104569170B
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: 2016-11-23
Anticipated expiration: 2033-10-29
Also published as: CN104569170A

Abstract

The invention provides the detection method of a kind of ginkgo-dipyridamine injection, the method includes finger printing test and the assay content of ginkgo-dipyridamine injection；Wherein finger printing test is the test of the finger printing to flavonoid in Folium Ginkgo extract and terpene lactone；Assay is the mensuration to flavonoid in Folium Ginkgo extract and terpene lactone；Compared with prior art, detection method is to the quality control of ginkgo-dipyridamine injection product and the discriminating of effective ingredient monomer and assay more effectively, method precision, stability and repeatability are the best, are the another excellent guarantees to this kind therapeutic effect.

Description

A kind of detection method of ginkgo-dipyridamine injection

Technical field:

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

Technical background:

Most common and harm in cardiovascular and cerebrovascular disease such as coronary heart disease, cerebral thrombosis, hypertension, the cerebral infarction Deng Junshi world today One of maximum disease, is also commonly encountered diseases, the frequently-occurring disease that harm our people is healthy, it has also become the main cause of human mortality it One；It was reported, sickness rate in recent years has and increases trend year by year, and Er Qiezhong, adolescent patients are continuously increased.Ginkgo Leaf Extract and Dipyridamole Injection It is made with dipyridamole by Folium Ginkgo extract, records and rise national standard (first in chemical drugs provincial standard Volume), standard No.: WS-10001-(HD-0087)-2002.This product energy antiplatelet aggregation, belongs to coronary artery expansion medicine, suitable For preventing and treat coronary heart disease, thrombotic disease, wherein containing flavonoid, terpenoid, phenols, life in Folium Ginkgo extract Alkaloids isoreactivity composition, can play improvement cardiac muscle and brain tissue metabolism, increase coronary artery and brain vessel blood, improve cardiac muscle and The effect of the blood supplies such as cerebral tissue, for treatment cardiovascular and cerebrovascular disease such as coronary heart diseases and angina pectoris, arrhythmia, cerebral thrombosis, senile Dementia etc. has preferable curative effect, can improve immunity of organisms simultaneously, and resist the disease occurs.But pharmaceutical preparation have to protected On the basis of card constant product quality controllable safety, more new development that could not be short.

Document " efficient liquid-phase chromatograph finger print atlas of dual qualitative and dual quantitative similarity method evaluation Ginkgo Leaf Extract and Dipyridamole Injection " (grandson State is auspicious, chromatograph the 4th phase of volume 25, in July, 2007) one disclosed herein with double qualitative similarities S and S, double quantitative similarities C With P evaluate Ginkgo Leaf Extract and Dipyridamole Injection HPLC finger printing, with kaempferol, rutin, Quercetin, isorhamnetin, Ginkgolides a and B Being reference substance with C, wherein chromatographic condition is CenturySIL C18BDS post, mobile phase A: water (containing 1% acetic acid)；B: acetonitrile (contains 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, detects wavelength 265nm, column temperature 30.0 ± 0.15 DEG C, elution time 80min.Although this detection method can preferably control Ginkgo Leaf Extract and Dipyridamole Injection Quality, but assay method is complicated, and to the separation of other concrete effective ingredient such as bilobalide etc. of product, evaluation etc. The most incomplete.Document " research of Ginkgo Leaf Extract and Dipyridamole Injection finger printing " (Song Jingyan, scientific and technological information exploitation and economical volume 18 the 6th Phase, in July, 2008) finger printing to Folium Ginkgo extract and injection disclosed herein studies, with rutin as reference substance, Wherein chromatographic condition be octadecylsilane chemically bonded silica be filler, mobile phase A: acetonitrile；B:0.4% phosphoric acid solution (uses three second Amine regulation pH to 2.5).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, detects wavelength 360nm, column temperature 40.0 DEG C, elution time 80min.The method is only using rutin as comparison, to terpenes such as flavonoid and ginkalide A such as the Quercetins in Folium Ginkgo extract Class lactone can not well be demarcated and make a comment or criticism, and also cannot evaluate the content of its effective ingredient.In order to preferably control said preparation Quality, it is ensured that the safety of medication, more preferable Instructing manufacture, make technology controlling and process more strict rationally, enable a consumer to recognize comprehensively Know product quality, need constantly to study and find out be more beneficial for product quality detection, the most concrete effective ingredient kind and contain The method of amount.

Summary of the invention:

It is an object of the invention to provide the detection method of a kind of ginkgo-dipyridamine injection, this method is for following product The index of detection, the means of detection, technical method etc. are provided, in order to preferably control this system to relevant production, testing agency The quality of agent, it is ensured that the safety of medication, it is possible to preferably Instructing manufacture, makes controlling of production process more strict rationally, makes to disappear The person's of expense energy full appreciation product quality, targeted product is:

The injection being prepared from by Folium Ginkgo extract 20-100 weight portion, dipyridamole 2-10 parts by weight of composition, bag Include be directly used in drug administration by injection injection, need to dilute after for the concentrated solution for injection of intravenous drip, directly for intravenous drip Glucose intravenous infusion and sodium chloride intravenous infusion and the Injectable sterile for preparing with freeze-drying or spray drying method Powder and aseptic block.

Such as, described product can be commercially available Ginkgo Leaf Extract and Dipyridamole Injection, and specification is 5ml, 10ml, by Guizhou benefit one hundred system The production of 3 companies such as medicine, Shanxi general moral Pharmaceutical, Tonghua paddy red pharmacy；Can also be patent CN200510200678.5, title For: freeze-dried formulation prepared by the preparation method that " ginkgo Damo freeze-drying preparation and preparation method thereof " provides, it is also possible to be patent CN200810068921.6, entitled: " composite preparation of Folium Ginkgo extract and dipyridamole and preparation method thereof " provides The ejection preparation prepared of preparation method.

The present invention is configured so that

The detection method of ginkgo-dipyridamine injection, including herein below:

The finger printing test of ginkgo-dipyridamine injection, is characterized as master's including with Folium Ginkgo extract flavones ingredient Finger printing and based on the finger printing of Folium Ginkgo extract terpene lactone composition characteristics；

The assay of ginkgo-dipyridamine injection, is characterized as main content including with Folium Ginkgo extract flavones ingredient Measure, be embodied in the content of Quercetin, kaempferide, isorhamnetin；With with Folium Ginkgo extract terpene lactone composition characteristics It is main assay, is embodied in the content of Ginkgolide A. B. C and bilobalide.

Wherein, fingerprint atlas detection method includes following content:

A, high performance liquid chromatography test is used to be characterized as main finger printing with Folium Ginkgo extract flavones ingredient:

(1) preparation of need testing solution: taking ginkgo-dipyridamine injection to be measured appropriate, injection volatilizes moisture, lyophilized powder It is placed directly within the mixed liquor of methanol-25% hydrochloric acid solution, lets cool after being heated to reflux, after filtration, take subsequent filtrate molten as test sample Liquid；

(2) preparation of reference substance solution: take the main active reference substance in appropriate Folium Ginkgo medical material, including rutin, Kaempferol rutinoside, isorhamnetin rutinoside, kaempferide, Quercetin, isorhamnetin, dissolve with water or methanol or ethanol, It is settled to suitable concn, as reference substance solution；

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

(4) formulation of standard finger-print: method described above is as formulating with Folium Ginkgo extract flavones ingredient feature It it is the means of testing of main finger printing；According to the collection of illustrative plates measured by 10 batches or more than 10 batches test samples, formulate standard fingerprint figure Spectrum, on the basis of the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, described In standard finger-print, total peak has 15～30；

(5) become with Folium Ginkgo extract flavonoid in using (1)～(3) described method as ginkgo-dipyridamine injection to be measured Divide the means of testing being characterized as main finger printing, prepare the finger printing of testing sample；

(6) finger printing of ginkgo-dipyridamine injection to be measured is contrasted with above-mentioned standard finger-print, should meet following Require is part or all of:

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

In ginkgo-dipyridamine injection finger printing the most to be measured, non-shared peak area must not exceed the 10% of total peak area；

Ginkgo-dipyridamine injection finger printing the most to be measured has ratio and the standard of the total peak area of 5%～50% In finger printing, relatively, its difference cannot be greater than ± 50% to the odds ratio of each total peak area；

B, use high performance liquid chromatography test based on the finger printing of Folium Ginkgo extract terpene lactone composition characteristics:

(1) preparation of need testing solution: taking ginkgo-dipyridamine injection to be measured appropriate, lyophilized powder is dissolved in water, injection Directly add 1%～4% hydrochloric acid solution, be extracted with ethyl acetate 3-4 time, divide and take acetic acid ethyl fluid, wash by 1%～10% sodium acetate solution Wash, divide and take sodium acetate liquid, then wash by ethyl acetate, divide and take ethyl acetate washings, wash with water 1-2 time, point water intaking washing liquid, use Ethyl acetate is washed, combined ethyl acetate liquid, reclaims ethyl acetate the most dry, and residue adds methanol makes dissolving, shakes up, as test sample Solution；

(2) preparation of reference substance solution: take ginkalide A, B, C and bilobalide appropriate, dissolves with methanol or ethanol, fixed Hold to suitable concn, as reference substance solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler: mobile phase A is methanol, flow phase B is oxolane: the solution of water=5-15:80-70, gradient elution, and flow velocity is 0.7～0.9ml/min, evaporative light scattering detection Device detects, and column temperature is in the range of 25～35 DEG C, and the record time is 60-70min；

(4) formulation of standard finger-print: method described above is as formulating with Folium Ginkgo extract terpene lactone constituents It is characterized as the means of testing of main finger printing；According to the collection of illustrative plates measured by 10 batches or more than 10 batches test samples, formulation standard refers to Stricture of vagina collection of illustrative plates, on the basis of the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, In described standard finger-print, total peak has 3～20；

(5) with Folium Ginkgo extract terpene lactone in using (1)～(3) described method as ginkgo-dipyridamine injection to be measured Constituents is characterized as the means of testing of main finger printing, prepares the finger printing of testing sample；

Ginkgo-dipyridamine injection finger printing the most to be measured has ratio and the mark of the total peak area of 30%～80% In quasi-finger printing, relatively, its difference cannot be greater than ± 50% to the odds ratio of each total peak area.

It is preferably:

(1) preparation of need testing solution: it is appropriate that precision weighs ginkgo-dipyridamine injection to be measured, and wherein injection is 10ml, lyophilized powder is the amount containing dipyridamole 4mg, injection volatilize after or lyophilized powder directly put methanol-25% hydrochloric acid solution (4: 1) mixed solution 25ml, is heated to reflux 30 minutes, lets cool, and filters and is transferred in 25ml measuring bottle, adds methanol to graduation mark, shakes up, As need testing solution；

(2) preparation of reference substance solution: precision weighs rutin, kaempferol rutinoside, isorhamnetin rutinoside, Rhizoma Kaempferiae Element, Quercetin, isorhamnetin, add methanol dissolve be respectively prepared every 1ml containing 0.03mg rutin, 0.02mg kaempferol rutinoside, 0.02mg isorhamnetin rutinoside, 0.03mg kaempferide, 0.03mg Quercetin, the solution of 0.02mg isorhamnetin, as right According to product solution；

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

Table 1 ginkgo-dipyridamine injection flavones ingredient gradient table

(4) formulation of standard finger-print: method described above is as formulating with Folium Ginkgo extract flavones ingredient feature It it is the means of testing of main finger printing；According to the collection of illustrative plates measured by 10 batches or more than 10 batches test samples, formulate standard fingerprint figure Spectrum, on the basis of the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, described In standard finger-print, total peak has 22；

(6) finger printing of ginkgo-dipyridamine injection to be measured is contrasted with above-mentioned standard finger-print, should meet following Require:

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

In ginkgo-dipyridamine injection finger printing the most to be measured, non-shared peak area must not exceed the 5% of total peak area；

Ginkgo-dipyridamine injection finger printing the most to be measured has ratio and the standard of the total peak area of 5%～50% In finger printing, relatively, its difference cannot be greater than ± 30% to the odds ratio of each total peak area；

(1) preparation of need testing solution: take ginkgo-dipyridamine injection to be measured appropriate, lyophilized powder 0.8mg Han dipyridamole Amount add 20ml water dissolution, injection 20ml directly adds 2% hydrochloric acid solution 2, with ethyl acetate shaking extract 4 times, each acetic acid The consumption of ethyl ester is respectively 15,10,10,10ml, point take acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divide and take acetic acid Sodium liquid, then wash by ethyl acetate 10ml, divide and take ethyl acetate washings, wash with water 2 times, each 20ml, point water intaking washing liquid, use Ethyl acetate 10ml is washed, combined ethyl acetate liquid, reclaims ethyl acetate the most dry, and residue adds methanol to be made dissolving and is transferred to 5ml In measuring bottle, add methanol to scale, shake up, as need testing solution；

(2) preparation of reference substance solution: take ginkalide A, B, C and bilobalide appropriate, accurately weighed, add methanol and dissolve It is respectively prepared every 1ml and contains 1mg ginkalide A, 1mg ginkalide B, 1mg ginkalide C, the solution of 2mg bilobalide, as Reference substance solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler: mobile phase A is methanol, flow phase B is oxolane: the solution of water=10:75, and according to the form below regulation carries out gradient elution, and flow velocity is 0.8ml/min, dissipates with evaporative light Penetrating detector detection, column temperature is at 30 DEG C, and the record time is 70min；Number of theoretical plate is calculated by bilobalide peak should be not less than 2500；

Table 2 ginkgo-dipyridamine injection terpene lactone composition gradient table

(4) formulation of standard finger-print: method described above is as formulating with Folium Ginkgo extract terpene lactone constituents It is characterized as the means of testing of main finger printing；According to the collection of illustrative plates measured by 10 batches or more than 10 batches test samples, formulation standard refers to Stricture of vagina collection of illustrative plates, on the basis of the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, In described standard finger-print, total peak has 7；

Ginkgo-dipyridamine injection finger printing the most to be measured has ratio and the mark of the total peak area of 30%～80% In quasi-finger printing, relatively, its difference cannot be greater than ± 30% to the odds ratio of each total peak area.

Wherein, content assaying method includes 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 uses octadecylsilane chemically bonded silica to be filler: mobile phase A is acetonitrile, flow phase B is 0.1%～2% phosphoric acid solution, gradient elution, and flow velocity is 0.9～1.1ml/min, detection wavelength is 254～360nm, column temperature For 30-40 DEG C, the record time is 80-90min；

(4) algoscopy: precision draws reference substance solution and need testing solution respectively, injects chromatograph of liquid, is measured；

(5) testing result: quercetin content in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection or every bottle of ginkgo-dipyridamole for injection Must not be less than 0.099mg, kaempferide content must not be less than 0.166mg, and isorhamnetin content must not be less than 0.044mg.

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

(5) testing result: in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection or every bottle of ginkgo-dipyridamole for injection, ginkalide A contains Amount must not be less than 0.057mg, and ginkalide B content must not be less than 0.021mg, and ginkalide C content must not be less than 0.037mg, Content of bilobalide must not be less than 0.081mg.

It is preferably:

(1) preparation of need testing solution: it is appropriate that precision weighs ginkgo-dipyridamine injection to be measured, after injection 10ml volatilizes Or lyophilized powder directly puts methanol-25% hydrochloric acid solution (4:1) mixed solution 25ml containing the amount of dipyridamole 4mg, it is heated to reflux 30 points Clock, lets cool, and filters and is transferred in 25ml measuring bottle, adds methanol to graduation mark, shakes up, as need testing solution；

Table 3 ginkgo-dipyridamine injection flavones ingredient gradient table

(4) algoscopy: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, enters Row measures；

(5) testing result: quercetin content in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection or every bottle of ginkgo-dipyridamole for injection Must not be less than 0.105mg, kaempferide content must not be less than 0.177mg, and isorhamnetin content must not be less than 0.048mg.

Table 4 ginkgo-dipyridamine injection terpene lactone composition gradient table

(4) algoscopy: precision draws reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid, enters Row measures；

(5) testing result: in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection or every bottle of ginkgo-dipyridamole for injection, ginkalide A contains Amount must not be less than 0.061mg, and ginkalide B content must not be less than 0.022mg, and ginkalide C content must not be less than 0.040mg, Content of bilobalide must not be less than 0.087mg.

Compared with prior art, the control ginkgo-dipyridamine injection product quality that the present invention can be more perfect.Due to this In product, the effective ingredient of Folium Ginkgo extract is complicated, if only with wherein one, two kind of composition its inherent quality is described, have 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 On, increase the matter comprehensively controlling ejection preparation based on the finger printing of Folium Ginkgo extract composition characteristics and assay thereof Amount is more effectively；Owing to having Folium Ginkgo extract and two kinds of compositions of dipyridamole in ginkgo-dipyridamine injection, it is the most effective Composition has certain interference each other, uses conventional liquid phase chromatogram condition, and the separating effect at each composition characteristics peak is difficult to full Foot requirement, only uses the condition of the present invention, selects suitable parameter to carry out gradient elution, just can obtain exclusive, accurate, steady Fixed, workable preferable detection method, is more controlled the content limit of concrete effective ingredient.Demonstrate,proved by experiment Bright, the quality control of the detection method ejection preparation product to making with Folium Ginkgo extract, dipyridamole more has Effect, method precision, stability are the highest.

Experimental example 1: be characterized as the research of main finger printing with Folium Ginkgo extract flavones ingredient

A, experimental apparatus, reagent and sample:

Rutin (0080-9705), kaempferol rutinoside (17650-84-9), isorhamnetin rutinoside (604-80- 8), kaempferide (110861-200808), Quercetin (10081-200406), isorhamnetin (110860-200608) reference substance are purchased Institute is supervised in Guiyang medicine；Acetonitrile, methanol and oxolane are chromatographically pure, and remaining reagent is analytical pure；Test ginkgo bilobate extract Injection is provided by Guizhou benefit one hundred company limited.

B, chromatographic condition are tested with system suitability:

1. the selection of the phase that flows:

Research process has been investigated (1) methanol 0.1%-2% phosphoric acid water, (2) acetonitrile--0.1%-2% phosphoric acid water, (3) respectively Acetonitrile--three kinds of flow phase system of 0.1%-2% acetic acid water, test through gradient or isocratic elution and compare.Result display flowing phase (1) Under the conditions of, peak shape is preferable, goes out peak and completely and is evenly distributed, and 6 the composition chromatographic peaks detected can reach baseline separation, contracts again Short analysis time, good separating effect；Under the conditions of flowing phase (2), peak shape is preferable, but appearance time is longer, goes out peak relatively in 1 hour Few, many components go out peak after being trapped in, and separate not exclusively；Under the conditions of flowing phase (3), peak shape is poor, separates bad；So stream Dynamic phase (1) is the most selected, wherein methanol 0.4% phosphoric acid water best results.

2. the selection of chromatographic column:

In research process, select the liquid-phase chromatographic column that octadecylsilane chemically bonded silica is filler of routine, respectively Having tried out SHIMADZU post, DIKMA post, Agela post and the chromatographic column of four kinds of trades mark of Agilent post, result shows four kinds of trades mark Chromatographic column peak shape have different, SHIMADZU post to differ greatly with DIKMA post, Agela post, Agilent post, Agela post Peak shape preferable, go out peak retention time and differ greatly, separating effect Agela post is than HIMADZU post and DIKMA post, Agilent post Good, therefore determine that Agela post is as fingerprint map analyzing post.

3. the investigation of column temperature

Chromatographic condition determines according to above-mentioned, column temperature is set to 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C respectively and investigates, when column temperature is every Raising 5 DEG C, appearance time reach 2min, peak integral translation, but peak shape is constant, when only 40 DEG C, peak shape has and necessarily moves down, explanation Ruggedness is good, does not produce impact to analyzing, and under 35 DEG C of column temperatures, baseline is more steady, therefore selects 35 DEG C of column temperatures.

4. the investigation of flow velocity

Chromatographic condition determines according to above-mentioned, is set to flow velocity respectively carry out for 0.9ml/min, 1.0ml/min, 1.1ml/min Investigating, the change of flow velocity only have impact on appearance time, but peak shape is basically identical, illustrates that the ruggedness of chromatographic condition is good, 0.9ml/ Min chromatogram baseline has and necessarily moves down, and 1.1ml/min flow velocity is relatively big to pillar pressure, therefore finally determines that flow velocity is 1.0ml/ min。

5. the determination of detection wavelength:

In research under the conditions of the flowing mutually of methanol-0.4% phosphoric acid water, investigated respectively different-waveband typical wavelengths 215, 254,280 the chromatographic peak situation, under 360nm, result shows, under 215nm, peak shape and baseline are unstable, with 254,280, Appearance time peak shape under 360nm is inconsistent, and 254, baseline is good under 280nm, 360nm, appearance time is basically identical, but 254, Under 280nm, peak shape is less, is not easy to observation analysis, and 360nm is mainly the maximum absorption wavelength of flavone, therefore final choice 360nm As detection wavelength.

6. record the selection of time

Prepare need testing solution by selected method, draw 10 μ L, inject chromatograph of liquid, by gradient condition, survey in accordance with the law Fixed, record 120min chromatogram.Chromatogram occurs without cutting edge of a knife or a sword shape after 75min, goes 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:

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

Eluent gradient:

Table 5 ginkgo-dipyridamine injection flavones ingredient gradient table

8. the preparation of need testing solution:

Take Ginkgo Leaf Extract and Dipyridamole Injection 10ml, volatilize rearmounted methanol-25% hydrochloric acid solution (4:1) mixed solution 25ml, heat back Flow 30 minutes, let cool, filter and be transferred in 25ml measuring bottle, add methanol to graduation mark, shake up, to obtain final product.

9. the preparation of object of reference solution:

Quercetin is one of Folium Ginkgo extract main active, and proportion is relatively in finger printing for its integral area Greatly and the most stable, take into account the research of intermediate and crude drug simultaneously, therefore select Quercetin as object of reference, and use methanol Making solvent, measurement result shows that the method is feasible.

10. finger printing stability, precision, repeated experiment

10.1 stability: take need testing solution, sample 10 μ L (0h, 2h, 4h, 8h, 12h) in different time, inject liquid phase Chromatograph, measures in accordance with the law.Result shows, each main chromatographic peak relative retention time and peak area account for total peak area 5% with superiors's Peak area ratio has no significant change (determining 19 common peaks), and its RSD is respectively 0.01%-0.22% and 0.26%-2.27%, symbol Close fingerprint pattern technology and require (RSD is all within 3%), have good stability.And through " chromatographic fingerprints of Chinese materia medica similarity is commented Valency system A version " calculate after, its similarity reaches more than 0.99.

10.2 precision: take need testing solution, the accurate 10 μ L that draw, injection chromatograph of liquid, repeat sample introduction 6 times, record Finger printing.Result shows, each main chromatographic peak relative retention time and peak area account for the total peak area 5% peak area with superiors Ratio has no significant change (determining 19 common peaks), and its RSD is respectively 0.01%-0.19% and 0.33%-2.11%, meets fingerprint Graphical spectrum technology requires (RSD is all within 3%), and precision is good.And through " similarity evaluation A Version " calculate after, its similarity reaches more than 0.99.

10.3 repeatability: take the test sample 6 parts of same lot number, be prepared as stated above and detect, investigate each Chromatographic peak relative time, the concordance of peak area ratio, result shows, each main chromatographic peak relative retention time and account for face, total peak The relative peak area of long-pending more than 5% chromatographic peak has no significant change (determining 19 common peaks), and its RSD is respectively 0.02%-0.36% And 0.60%-2.59%, meeting fingerprint pattern technology requirement (RSD is all within 3%), repeatability is good.And through " Chinese medicine chromatograph Fingerprint similarity evaluate system A version " calculate after, its similarity reaches more than 0.99.

11. finger printing and the formulation of technical parameter:

11.1 reference fingerprints: according to 10 batches of Ginkgo Leaf Extract and Dipyridamole Injection sample finger printing, with Quercetin reference substance be Object of reference, formulates standard finger-print, is compared with standard finger-print by test sample finger printing, similarity all 0.95～ Between 1.00.

11.2 demarcation having fingerprint peaks

According to the testing result of 10 batches of Ginkgo Leaf Extract and Dipyridamole Injection finger printing, see Fig. 1.Have 22 peaks all can preferably divide From, 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 Peak all occurs in 10 batches of Ginkgo Leaf Extract and Dipyridamole Injection at identical relative retention time, and its finger printing area sum accounts for total amount It is all higher than 95%, thus demarcates these 22 chromatographic peaks for total fingerprint peaks.Wherein 7 colors are pointed out by reference substance Comparability test Spectral peak, peak 5,10,11, S, 17,18,20 are followed successively by rutin, kaempferol rutinoside, isorhamnetin rutinoside, Cortex querci dentatae respectively Element, reaches not, kaempferide, isorhamnetin.

11.3 peak area ratios having fingerprint peaks

5,10, No. 11 peaks (rutin, kaempferol rutinoside, isorhamnetin rutinoside) are that first three ten minutes unit goes out peak Area accounts for the total peak area total peak more than 5% less than 10%, and its difference is respectively+11%-14% ,+13%-22% ,+13%-16%, symbol Close " technology of Chinese medicine finger printing research requires " (provisional) and cannot be greater than positive and negative 30%.S, No. 18 peaks (Quercetin, mountains How element) it is that latter 30 minutes units go out peak area and account for the total peak area total peak more than 10%, its difference is respectively+12%-14% ,+ 9%-12%, meets " technology of Chinese medicine finger printing research requires " (provisional) and cannot be greater than positive and negative 25%.Remaining first three ten Minute total peak unit peak area accounts for total peak area and is below 5%, and it is equal that latter 30 minutes total peak unit peak areas account for total peak area Less than 10%, according to " technology of Chinese medicine finger printing research requires " (provisional), its peak area ratio is not required.

11.4 non-shared peak area

10 batch Ginkgo Leaf Extract and Dipyridamole Injection respective HPLC spectrogram measurement results show, non-shared peak area accounts for face, total peak Long-pending less than 5%, meet " technology of Chinese medicine finger printing research requires " (provisional).

Experimental example 2 is based on the research of the finger printing of Folium Ginkgo extract terpene lactone composition characteristics

A, experimental apparatus, reagent and sample:

Ginkalide A (110862-200305), ginkalide B (110863-200305), ginkalide C (110864- 200), bilobalide (110865-200404) reference substance is purchased from Guiyang medicine prison institute；Acetonitrile, methanol and oxolane are chromatograph Pure, remaining reagent is analytical pure；Test Ginkgo Leaf Extract and Dipyridamole Injection is provided by Guizhou benefit one hundred company limited.

B, chromatographic condition are tested with system suitability:

1. the selection of the phase that flows:

Research process investigates (1) methanol respectively: (oxolane: water), (2) acetonitrile: (oxolane: water), (3) are different Propanol: methanol: three kinds of flow phase system of water, tests through gradient or isocratic elution and compares.Under the conditions of result display flowing phase (1), Peak shape is preferable, goes out peak and completely and is evenly distributed, and 4 the composition chromatographic peaks detected can reach baseline separation, shortens again point The analysis time, good separating effect；Under the conditions of flowing phase (2), peak shape is preferable, but appearance time is longer；Under the conditions of flowing phase (3), peak Shape is poor, separates bad；So flowing phase (1) is the most selected, wherein oxolane: water=10:75 best results.

2. the selection of chromatographic column:

In research process, using Kromasil post, DIKMA post, Agela post respectively, Agilent post is analyzed, and compares point From result, four kinds of chromatographic columns compare, and peak shape has different, DIKMA post and Kromasil post, Agela post, Agilent post Differing greatly, the peak shape of Agela post is preferable, goes out peak retention time and differs greatly, separating effect Agela post than HIMADZU post with DIKMA post, Agilent post are good, therefore determine that Agela post is as fingerprint map analyzing post.

3. the investigation of column temperature

Chromatographic condition determines according to above-mentioned, column temperature is set to 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C respectively and investigates, when column temperature is every Raise 5 DEG C, appearance time reach 1min, peak integral translation, but peak shape is constant, and 25 DEG C of column temperature base lines are unstable, when column temperature arrives When 35 DEG C and 40 DEG C, peak shape has and necessarily moves down, and general description ruggedness is good, does not produce impact to analyzing, therefore selects 30 DEG C of column temperatures.

4. the investigation of flow velocity

Flow velocity is set to by research process respectively investigate for 0.7ml/min, 0.8ml/min, 0.9ml/min, flow velocity Change only have impact on appearance time, but peak shape is basically identical, illustrates that the ruggedness of chromatographic condition is good, 0.7ml/min with The chromatogram baseline of 0.9ml/min flow velocity is unstable compared with 0.8ml/min, therefore finally determines that flow velocity is 0.8ml/min.

5. record the selection of time

Prepare need testing solution by selected method, draw 20 μ L, inject chromatograph of liquid, by gradient condition, survey in accordance with the law Fixed, record 60min chromatogram.Chromatogram occurs without cutting edge of a knife or a sword shape after 50min, goes 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:

Flowing phase: methanol: (oxolane: water) (10:75) (gradient), chromatographic column is Agela post, and column temperature is 30 DEG C, stream Speed 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:

Taking Ginkgo Leaf Extract and Dipyridamole Injection 20ml, precision measures, and adds 2% hydrochloric acid solution 2, extracts 4 times with ethyl acetate shaking (15,10,10,10ml), point take acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divide and take sodium acetate liquid, then use acetic acid second Ester 10ml washs.Divide and take ethyl acetate washing liquid, wash with water 2 times, each 20ml, point water intaking washing liquid, wash by ethyl acetate 10ml Washing, merge ethyl acetate liquid, recovery of acetic acid ethyl ester is to dry, and residue methanol dissolves and is transferred in 5ml measuring bottle, adds methanol to carving Degree, shakes up, to obtain final product.

8. the preparation of object of reference solution:

Bilobalide is one of Folium Ginkgo extract lactone active component, and its integral area is institute's accounting in finger printing Example more greatly and the most stable, takes into account the research of intermediate and crude drug simultaneously, therefore selectes bilobalide as object of reference, and With methanol as solvent, measurement result shows that the method is feasible.

9. finger printing stability, precision, repeated experiment

9.1 stability: take need testing solution, sample 20 μ L (0h, 1h, 4h, 8h, 12h) in different time, inject liquid phase color Spectrometer, measures in accordance with the law.Result shows, each main chromatographic peak relative retention time and peak area account for the total peak area 5% peak with superiors Area ratio has no significant change (determining 7 common peaks), and its RSD is respectively 0.23%-0.44% and 0.43%-1.83%, meets Fingerprint pattern technology requires (RSD is all within 3%), has good stability.And through " chromatographic fingerprints of Chinese materia medica similarity evaluation System A version " calculate after, its similarity reaches more than 0.99.

9.2 precision: taking need testing solution, the accurate 20 μ L that draw, injection chromatograph of liquid, repeat sample introduction 6 times, record refers to Stricture of vagina collection of illustrative plates.Result shows, each main chromatographic peak relative retention time and peak area account for the total peak area 5% peak area ratio with superiors Value has no significant change (determining 7 common peaks), and its RSD is respectively 0.18%-0.40% and 0.28%-2.08%, meets fingerprint image Spectral technology requires (RSD is all within 3%), and precision is good.And through " similarity evaluation A Version " calculate after, its similarity reaches more than 0.99.

9.3 repeatability: take the test sample 6 parts of same lot number, be prepared as stated above and detect, investigate colors Spectral peak relative time, the concordance of peak area ratio, result shows, each main chromatographic peak relative retention time and account for total peak area The relative peak area of more than 5% chromatographic peak has no significant change (determining 7 common peaks), its RSD be 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 through " Chinese medicine chromatograph refers to Stricture of vagina collection of illustrative plates similarity evaluation system A version " calculate after, its similarity reaches more than 0.99.

10. finger printing and the formulation of technical parameter:

10.1 reference fingerprints: according to 10 batches of Ginkgo Leaf Extract and Dipyridamole Injection sample finger printing, with bilobalide reference substance For object of reference, formulate standard finger-print, test sample finger printing compared with standard finger-print, similarity all 0.95～ Between 1.00.

10.2 demarcation having fingerprint peaks

According to the testing result of 10 batches of Ginkgo Leaf Extract and Dipyridamole Injection finger printing, see Fig. 2.Have 7 peaks all can preferably separate, Be numbered respectively, wherein 1,2, S, 3,4,5, No. 6 peak in 10 batches of Ginkgo Leaf Extract and Dipyridamole Injection all identical when relatively retaining Between place occur, its finger printing area sum accounts for total amount and is all higher than 95%, thus demarcates these 7 chromatographic peaks for total fingerprint peaks.Its In pointed out 4 chromatographic peaks by reference substance Comparability test, peak 2, S, 3,4 are followed successively by ginkalide C, bilobalide, silver respectively Fructus Pruni lactone A, ginkalide B.

10.3 peak area ratios having fingerprint peaks

S peak (bilobalide) is that first three ten minutes unit goes out peak area and accounts for the total peak area total peak more than 20%, and it is poor Value is+10%-9%, meets " technology of Chinese medicine finger printing research requires " (provisional) and cannot be greater than positive and negative 20%.No. 1,2 Number peak (ginkalide C) is that first three ten minutes unit goes out peak area and accounts for the total peak area total peak more than or equal to 5% less than 10%, Its difference is respectively+17%-12% ,+17%-14%, meets " technology of Chinese medicine finger printing research requires " (provisional) no Must be more than positive and negative 30%.No. 3 peaks (ginkalide A) are that first three ten minutes unit goes out peak area and accounts for total peak area more than or equal to 10% Total peak less than 20%, its difference is+12%-15%, meets " technology of Chinese medicine finger printing research requires " (provisional) Cannot be greater than positive and negative 25%.After No. 6 peaks are, 30 minutes units go out peak area and account for the total peak area total peak more than 10%, its difference For+9%-8%, meet " technology of Chinese medicine finger printing research requires " (provisional) and cannot be greater than positive and negative 25%.Remaining first three Ten minutes total peak unit peak areas account for total peak area and are below 5%, and within latter 30 minutes, total peak unit peak area accounts for total peak area Being below 10%, according to " technology of Chinese medicine finger printing research requires " (provisional), its peak area ratio is not required.

10.4 non-shared peak area

Experimental example 3 ginkgo-dipyridamine injection assay is studied

1. instrument and reagent

1.1 instruments: analytical balance (prunus mume (sieb.) sieb.et zucc. Teller), nutsch filter, high performance liquid chromatograph (Agilent1100 type, band diode Array detector, evaporative light detector).

1.2 reagents: kaempferide (110861-200808), Quercetin (10081-200406), isorhamnetin (110860- 200608), ginkalide A (110862-200305), ginkalide B (110863-200305), ginkalide C (110864- 200), bilobalide (110865-200404) reference substance is purchased from Guiyang medicine prison institute；Acetonitrile, methanol and oxolane are chromatograph Pure, remaining reagent is analytical pure；Test Ginkgo Leaf Extract and Dipyridamole Injection is provided by Guizhou benefit one hundred company limited.

2. flavonoid multi-target ingredient assay research

2.1 chromatographic condition

Measure with Ginkgo Leaf Extract and Dipyridamole Injection finger printing flavonoid.

The preparation of 2.2 need testing solutions

2.3 linear relationship

Precision weighs Quercetin, kaempferide, isorhamnetin in right amount, adds methanol dissolving and makes mass concentration respectively The reference substance solution of 0.137mg/ml, 0.090mg/ml, 0.067mg/ml, injects HPLC by the chromatographic condition drafted and measures.Mongolian oak Skin element sample size is respectively 2,6,10,14,18 μ l, and kaempferide sample size is respectively 15,20,25,30,35 μ l, and isorhamnetin enters Sample amount is respectively 5,10,15,20,25 μ l.With peak area, sample introduction quality is carried out linear regression, obtain the recurrence side of each tested composition Journey, correlation coefficient and the range of linearity.

Table 7 flavonoids effective constituent linear relationship table

2.4 assay stability, precision, repeatability, the response rate are tested

Stability: prepare preparation need testing solution according to Ginkgo Leaf Extract and Dipyridamole 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 is stable in showing need testing solution 12h.Its RSD is respectively 1.15%, 0.83% and 0.97%, meets quality standard technology and requires (RSD is all within 3%), has good stability.

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

Repeatability: take the Ginkgo Leaf Extract and Dipyridamole Injection 6 parts of same lot number, prepare test sample according to need testing solution preparation method molten Liquid 10 μ l, measures respectively, calculates each index content, and RSD, between 1.10%-1.35%, meets quality standard technology and requires (RSD All within 3%), repeatability is good.

The response rate: use average recovery method, process sample by above-mentioned sample solution preparation method, adds appropriate Quercetin, kaempferide, isorhamnetin reference substance solution (0.161mg/ml, 0.401mg/ml, 0.155mg/ml), make low, in, High 3 kinds of variable concentrations need testing solutions, measure respectively, and average recovery rate is 100.13%, and RSD is 2.57%, meets quality standard Technology requires (RSD is all within 3%).

3. flavonoid multi-target ingredient assay research

3.1 chromatographic condition

Measure with Ginkgo Leaf Extract and Dipyridamole Injection finger printing lactone.

The preparation of 3.2 need testing solutions

Taking Ginkgo Leaf Extract and Dipyridamole Injection about 20ml, precision measures, and adds 2% hydrochloric acid solution 2, extracts 4 times with ethyl acetate shaking (15,10,10,10ml), point take acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divide and take sodium acetate liquid, then use acetic acid second Ester 10ml washs.Divide and take ethyl acetate washing liquid, wash with water 2 times, each 20ml, point water intaking washing liquid, wash by ethyl acetate 10mL Washing, merge ethyl acetate liquid, recovery of acetic acid ethyl ester is to dry, and residue methanol dissolves and is transferred in 5ml measuring bottle, adds methanol to carving Degree, shakes up, to obtain final product.

3.3 linear relationship

Precision weighs Ginkgolide A. B. C and bilobalide is appropriate, adds methanol dissolving and makes mass concentration respectively 0.425mg/ml, 0.103mg/ml, 0.097mg/ml, the reference substance solution of 0.163mg/ml, injected by the chromatographic condition drafted HPLC measures.Ginkalide A sample size is respectively 1,3,5,7,9 μ l, and ginkalide 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 carries out linear regression to sample introduction quality, obtains regression equation, correlation coefficient and the range of linearity of each tested composition.

Table 8 terpene lactone effective ingredient linear relationship table

3.4 assay stability, precision, repeatability, the response rate are tested

Stability: prepare preparation need testing solution according to Ginkgo Leaf Extract and Dipyridamole 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 is stable in showing need testing solution 12h.Its RSD is respectively 0.56%, 0.82% and 1.83%, meets quality standard technology and requires (RSD is all within 3%), has good stability.

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

Repeatability: take the Ginkgo Leaf Extract and Dipyridamole Injection 6 parts of same lot number, prepare test sample according to need testing solution preparation method molten Liquid 20 μ l, measures respectively, calculates each index content, and RSD, between 0.49%-2.48%, meets quality standard technology and requires (RSD All within 3%), repeatability is good.

The response rate: use average recovery method, process sample by above-mentioned sample solution preparation method, adds appropriate Ginkgolide A. B. C and bilobalide reference substance solution (0.0198mg/ml, 0.0109mg/ml, 0.0201mg/ml, 0.0409mg/ml), making 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 technology and requires (RSD is all within 3%).

4. assay result

Take the Ginkgo Leaf Extract and Dipyridamole Injection of 10 batches respectively, the chromatographic condition drafted by need testing solution preparation method, survey The content of fixed 3 main components, measurement result is shown in Table.

Table 9 Ginkgo Leaf Extract and Dipyridamole Injection assay result (mg/ bottle (10ml))

Result shows, our company is Quercetin in 10 batch Ginkgo Leaf Extract and Dipyridamole Injection samples of production in 2009 to 2012 years Average out to 0.124mg/ bottle；Kaempferide average out to 0.208mg/ bottle；Isorhamnetin average out to 0.055mg/ bottle；Ginkalide A is put down It is 0.072mg/ bottle；Ginkalide B average out to 0.026mg/ bottle；Ginkalide C average out to 0.047mg/ bottle；Bilobalide is put down It is 0.102mg/ bottle.Comprehensive analysis 10 batch Ginkgo Leaf Extract and Dipyridamole Injection finished product measurement results, due to each batch Semen Ginkgo phyllogen medicine Material has different, and then has influence on the content of each effective ingredient in Ginkgo Leaf Extract and Dipyridamole Injection, therefore each composition measurement standard is fixed Being not less than 0.099mg/ bottle for Quercetin, kaempferide is not less than 0.166mg/ bottle, and isorhamnetin is not less than 0.044mg/ bottle, Semen Ginkgo Lactone A is not less than 0.057mg/ bottle, and ginkalide B is not less than 0.021mg/ bottle, and ginkalide C is not less than 0.037mg/ bottle, in vain Really lactone is not less than 0.081mg/ bottle.

Accompanying drawing explanation

Fig. 1 is that Ginkgo Leaf Extract and Dipyridamole Injection flavonoid finger printing of the present invention studies chromatogram；

Fig. 2 is that Ginkgo Leaf Extract and Dipyridamole Injection terpene lactone finger printing of the present invention studies chromatogram.

Detailed description of the invention

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

Embodiment 1: use in liquid chromatography for measuring Ginkgo Leaf Extract and Dipyridamole Injection special with Folium Ginkgo extract flavones ingredient Levying is main finger printing:

(1) preparation of need testing solution: precision weighs Ginkgo Leaf Extract and Dipyridamole Injection 10ml to be measured, volatilizes rearmounted methanol-25% salt Acid solution (4:1) mixed solution 25ml, is heated to reflux 30 minutes, lets cool, and filters and is transferred in 25ml measuring bottle, adds methanol to scale Line, shakes up, as need testing solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler；Mobile phase A is acetonitrile, and flow 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, detection wavelength is 360nm, Column temperature is 35 DEG C, and the record time is 90min, and number of theoretical plate is calculated by Quercetin peak should be not less than 2500；

(5) with Folium Ginkgo extract flavones ingredient in using (1)～(3) described method as Ginkgo Leaf Extract and Dipyridamole Injection to be measured It is characterized as the means of testing of main finger printing, prepares the finger printing of testing sample；

(6) Ginkgo Leaf Extract and Dipyridamole Injection finger printing to be measured is contrasted with above-mentioned standard finger-print, meets the following requirements:

I. calculate the finger printing of Ginkgo Leaf Extract and Dipyridamole Injection to be measured and the similarity of standard finger-print, should be 0.95～ 1.00；

In Ginkgo Leaf Extract and Dipyridamole Injection finger printing the most to be measured, non-shared peak area must not exceed the 5% of total peak area；

The ratio having the total peak area of 5%～50% in Ginkgo Leaf Extract and Dipyridamole Injection finger printing the most to be measured refers to standard In stricture of vagina collection of illustrative plates, relatively, its difference cannot be greater than ± 30% to the odds ratio of each total peak area.

Embodiment 2: use in liquid chromatography for measuring Ginkgo Leaf Extract and Dipyridamole Injection with Folium Ginkgo extract terpene lactone composition It is characterized as main finger printing:

(1) preparation of need testing solution: take Ginkgo Leaf Extract and Dipyridamole Injection 20ml to be measured, adds 2% hydrochloric acid solution 2, uses acetic acid second Ester shaking is extracted 4 times, the consumption of each ethyl acetate is respectively 15,10,10,10ml, point take acetic acid ethyl fluid, use 5% sodium acetate Solution 20ml washs, and divides and takes sodium acetate liquid, then washs by ethyl acetate 10ml, divides and takes ethyl acetate washings, washes with water 2 times, 20ml every time, point water intaking washing liquid, to wash by ethyl acetate 10ml, combined ethyl acetate liquid, recovery ethyl acetate is to dry, and residue adds Methanol makes dissolving and is transferred in 5ml measuring bottle, adds methanol to scale, shakes up, as need testing solution；

(3) chromatographic condition: mobile phase A is methanol, Mobile phase B is oxolane: 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, Detecting with evaporative light scattering detector, column temperature is at 30 DEG C, and the record time is 70min；Number of theoretical plate presses the calculating of bilobalide peak should It is not less than 2500；

(5) with Folium Ginkgo extract terpene lactone class in using (1)～(3) described method as Ginkgo Leaf Extract and Dipyridamole Injection to be measured Composition characteristics is the means of testing of main finger printing, prepares the finger printing of testing sample；

(6) finger printing of Ginkgo Leaf Extract and Dipyridamole Injection to be measured is contrasted with above-mentioned standard finger-print, meets the following requirements:

Ginkgo Leaf Extract and Dipyridamole Injection finger printing the most to be measured has ratio and the standard of the total peak area of 30%～80% In finger printing, relatively, its difference cannot be greater than ± 30% to the odds ratio of each total peak area.

Embodiment 3: use in liquid chromatography for measuring ginkgo-dipyridamole for injection special with Folium Ginkgo extract flavones ingredient Levying is main finger printing:

(1) preparation of need testing solution: it is the amount containing dipyridamole 4mg that precision weighs ginkgo Damo freeze-drying powder to be measured, directly Connect and put methanol-25% hydrochloric acid solution (4:1) mixed solution 25ml, be heated to reflux 30 minutes, let cool, filter and be transferred to 25ml measuring bottle In, add methanol to graduation mark, shake up, as need testing solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler；Mobile phase A is acetonitrile, and flow 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, detection wavelength is 240nm, Column temperature is 30 DEG C, and the record time is 80min；

(5) with Folium Ginkgo extract flavones ingredient in using (1)～(3) described method as ginkgo-dipyridamole for injection to be measured It is characterized as the means of testing of main finger printing, prepares the finger printing of testing sample；

(6) ginkgo-dipyridamole for injection finger printing to be measured is contrasted with above-mentioned standard finger-print, meets the following requirements:

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

In ginkgo-dipyridamole for injection finger printing the most to be measured, non-shared peak area is less than the 10% of total peak area；

Embodiment 4: use in liquid chromatography for measuring ginkgo-dipyridamole for injection with Folium Ginkgo extract terpene lactone composition It is characterized as main finger printing:

(1) preparation of need testing solution: taking ginkgo Damo freeze-drying powder to be measured is that the amount containing dipyridamole 0.8mg adds 20ml water Dissolve, add 2% hydrochloric acid solution 2, extract 4 times with ethyl acetate shaking, the consumption of each ethyl acetate is respectively 15,10,10, 10ml, divides and takes acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divides and takes sodium acetate liquid, then wash by ethyl acetate 10ml, Divide and take ethyl acetate washings, wash with water 2 times, each 20ml, point water intaking washing liquid, wash by ethyl acetate 10ml, merge acetic acid Ethyl ester liquid, reclaims ethyl acetate to dry, and residue adds methanol to be made dissolving and is transferred in 5ml measuring bottle, adds methanol to scale, shakes up, As need testing solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler；Mobile phase A is methanol, and flow phase B is oxolane: 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, detects with evaporative light scattering detector, and column temperature is at 25 DEG C, during record Between be 70min；

(5) with Folium Ginkgo extract terpene lactone class in using (1)～(3) described method as ginkgo-dipyridamole for injection to be measured Composition characteristics is the means of testing of main finger printing, prepares the finger printing of testing sample；

(6) finger printing of ginkgo-dipyridamole for injection to be measured is contrasted with above-mentioned standard finger-print, meets the following requirements:

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

The ratio having the total peak area of 30%～80% in ginkgo-dipyridamole for injection finger printing the most to be measured refers to standard In stricture of vagina collection of illustrative plates, relatively, its difference cannot be greater than ± 50% to the odds ratio of each total peak area.

Embodiment 5: the content of flavonoids effective constituent in employing liquid chromatography for measuring Ginkgo Leaf Extract and Dipyridamole Injection:

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler: mobile phase A is acetonitrile, flow 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, detection wavelength is 360nm, Column temperature is 35 DEG C, and the record time is 90min, and number of theoretical plate is calculated by Quercetin peak should be not less than 2500；

(5) testing result: in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection, quercetin content must not be less than 0.099mg, Rhizoma Kaempferiae Cellulose content must not be less than 0.166mg, and isorhamnetin content must not be less than 0.044mg.

Embodiment 6: the content of terpene lactone effective ingredient in employing liquid chromatography for measuring Ginkgo Leaf Extract and Dipyridamole Injection:

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler: mobile phase A is methanol, flow phase B is oxolane: 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, detects with evaporative light scattering detector, and column temperature, at 30 DEG C, records the time For 70min；Number of theoretical plate is calculated by bilobalide peak should be not less than 2500；

(5) testing result: in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection, ginkalide A content must not be less than 0.057mg, silver Fructus Pruni lactone B content must not be less than 0.021mg, and ginkalide C content must not be less than 0.037mg, and content of bilobalide must not be less than 0.081mg。

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

(1) preparation of need testing solution: precision weighs the ginkgo Damo freeze-drying powder to be measured amount containing dipyridamole 4mg, directly Put methanol-25% hydrochloric acid solution (4:1) mixed solution 25ml, be heated to reflux 30 minutes, let cool, filter and be transferred in 25ml measuring bottle, Add methanol to graduation mark, shake up, as need testing solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler: mobile phase A is acetonitrile, flow phase B is 0.1% 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, detection wavelength is 280nm, Column temperature is 40 DEG C, and the record time is 90min；

(5) testing result: quercetin content in this product every bottle ginkgo-dipyridamole for injection (specification: 4mg in terms of dipyridamole) Must not be less than 0.105mg, kaempferide content must not be less than 0.177mg, and isorhamnetin content must not be less than 0.048mg.

Embodiment 8: the content of terpene lactone effective ingredient in employing liquid chromatography for measuring ginkgo-dipyridamole for injection:

(1) preparation of need testing solution: taking ginkgo Damo freeze-drying powder to be measured is the amount containing dipyridamole 0.8mg, adds 20ml Water dissolution, adds 2% hydrochloric acid solution 2, extracts 4 times with ethyl acetate shaking, the consumption of each ethyl acetate is respectively 15,10, 10,10ml, divides and takes acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divides and takes sodium acetate liquid, then wash by ethyl acetate 10ml Wash, divide and take ethyl acetate washings, wash with water 2 times, each 20ml, point water intaking washing liquid, wash by ethyl acetate 10ml, merge second Acetoacetic ester liquid, reclaims ethyl acetate to dry, and residue adds methanol to be made dissolving and is transferred in 5ml measuring bottle, adds methanol to scale, shakes Even, as need testing solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler: mobile phase A is methanol, flow phase B is oxolane: the solution of water=5:80, gradient elution, and order is 0-20min, 75%-75%B；20-60min, 75%-32%B； 60-70min, 32%-75%B；Flow velocity is 0.7ml/min, detects with evaporative light scattering detector, and column temperature, at 35 DEG C, records the time For 70min；

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

Claims

1. a detection method for ginkgo-dipyridamine injection, the method includes herein below: with Folium Ginkgo extract flavonoid Composition characteristics is main finger printing test and tests based on the finger printing of Folium Ginkgo extract terpene lactone composition characteristics And based on Folium Ginkgo extract flavones ingredient Quercetin, kaempferide, the assay of isorhamnetin and carry with Folium Ginkgo Take thing terpene lactone composition Ginkgolide A. B. C and bilobalide is main assay；It is characterized in that:

The concrete detecting step of determining fingerprint pattern is:

A, the test of high liquid chromatography method is used to be characterized as main finger printing with Folium Ginkgo extract flavones ingredient:

(1) preparation of need testing solution: taking ginkgo-dipyridamine injection to be measured appropriate, injection volatilizes moisture, and lyophilized powder is direct It is placed in the mixed liquor of methanol-25% hydrochloric acid solution, lets cool after being heated to reflux, after filtration, take subsequent filtrate as need testing solution；

(2) preparation of reference substance solution: take the main active reference substance in appropriate Folium Ginkgo medical material, including rutin, Rhizoma Kaempferiae Phenol rutinoside, isorhamnetin rutinoside, kaempferide, Quercetin, isorhamnetin, dissolve with water or methanol or ethanol, constant volume To suitable concn, as reference substance solution；

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

(4) formulation of standard finger-print: method described above is characterized as master as formulating with Folium Ginkgo extract flavones ingredient The means of testing of finger printing；According to the collection of illustrative plates measured by 10 batches or more than 10 batches test samples, formulate standard finger-print, with On the basis of the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, described standard In finger printing, total peak has 15～30；

(5) with Folium Ginkgo extract flavones ingredient spy in using (1)～(3) described method as ginkgo-dipyridamine injection to be measured Levy the means of testing of the finger printing being master, prepare the finger printing of testing sample；

(6) finger printing of ginkgo-dipyridamine injection to be measured is contrasted with above-mentioned standard finger-print, should meet the following requirements Part or all of:

Ginkgo-dipyridamine injection finger printing the most to be measured has ratio and the standard fingerprint of the total peak area of 5%～50% In collection of illustrative plates, relatively, its difference cannot be greater than ± 50% to the odds ratio of each total peak area；

(1) preparation of need testing solution: taking ginkgo-dipyridamine injection to be measured appropriate, lyophilized powder is dissolved in water, and injection is direct Add 1%～4% hydrochloric acid solution, be extracted with ethyl acetate 3-4 time, divide and take acetic acid ethyl fluid, wash by 1%～10% sodium acetate solution Wash, divide and take sodium acetate liquid, then wash by ethyl acetate, divide and take ethyl acetate washings, wash with water 1-2 time, point water intaking washing liquid, use Ethyl acetate is washed, combined ethyl acetate liquid, reclaims ethyl acetate the most dry, and residue adds methanol makes dissolving, shakes up, as test sample Solution；

(2) preparation of reference substance solution: take ginkalide A, B, C and bilobalide appropriate, dissolves with methanol or ethanol, is settled to Suitable concn, as reference substance solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler: mobile phase A is methanol, and Mobile phase B is Oxolane: the solution of water=5-15:80-70, gradient elution, flow velocity is 0.7～0.9ml/min, evaporative light scattering detector Detection, column temperature is in the range of 25～35 DEG C, and the record time is 60-70min；

(4) formulation of standard finger-print: method described above is as formulating with Folium Ginkgo extract terpene lactone constituents feature It it is the means of testing of main finger printing；According to the collection of illustrative plates measured by 10 batches or more than 10 batches test samples, formulate standard fingerprint figure Spectrum, on the basis of the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, described In standard finger-print, total peak has 3～20；

(5) become with Folium Ginkgo extract terpene lactone class in using (1)～(3) described method as ginkgo-dipyridamine injection to be measured Divide the means of testing being characterized as main finger printing, prepare the finger printing of testing sample；

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

The ratio having the total peak area of 30%～80% in ginkgo-dipyridamine injection finger printing the most to be measured refers to standard In stricture of vagina collection of illustrative plates, relatively, its difference cannot be greater than ± 50% to the odds ratio of each total peak area；

The concrete detecting step of assay is:

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

(5) testing result: in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection or every bottle of ginkgo-dipyridamole for injection, quercetin content must not Less than 0.099mg, kaempferide content must not be less than 0.166mg, and isorhamnetin content must not be less than 0.044mg；

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

(5) testing result: ginkalide A content in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection or every bottle of ginkgo-dipyridamole for injection Must not be less than 0.057mg, ginkalide B content must not be less than 0.021mg, and ginkalide C content must not be less than 0.037mg, in vain Really lactone content must not be less than 0.081mg.

2. according to the detection method of the ginkgo-dipyridamine injection described in claim 1, it is characterised in that determining fingerprint pattern walks Suddenly following content is included:

(1) preparation of need testing solution: it is appropriate that precision weighs ginkgo-dipyridamine injection to be measured, and wherein injection is 10ml, freezes Dry powder is the amount containing dipyridamole 4mg, and injection volatilizes afterwards or lyophilized powder directly puts methanol-25% hydrochloric acid solution mixed solution 25ml, is heated to reflux 30 minutes, lets cool, and filters and is transferred in 25ml measuring bottle, adds methanol to graduation mark, shakes up, as test sample Solution；

(2) preparation of reference substance solution: precision weigh rutin, kaempferol rutinoside, isorhamnetin rutinoside, kaempferide, Quercetin, isorhamnetin, add methanol dissolve be respectively prepared every 1ml containing 0.03mg rutin, 0.02mg kaempferol rutinoside, 0.02mg isorhamnetin rutinoside, 0.03mg kaempferide, 0.03mg Quercetin, the solution of 0.02mg isorhamnetin, as right According to product solution；

(3) chromatographic condition: chromatographic column uses 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, detection Wavelength is 360nm, column temperature is 35 DEG C, and the record time is 90min, and number of theoretical plate is calculated by Quercetin peak should be not less than 2500；

(4) formulation of standard finger-print: method described above is characterized as master as formulating with Folium Ginkgo extract flavones ingredient The means of testing of finger printing；According to the collection of illustrative plates measured by 10 batches or more than 10 batches test samples, formulate standard finger-print, with On the basis of the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, described standard In finger printing, total peak has 22；

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

Ginkgo-dipyridamine injection finger printing the most to be measured has ratio and the standard fingerprint of the total peak area of 5%～50% In collection of illustrative plates, relatively, its difference cannot be greater than ± 30% to the odds ratio of each total peak area；

(1) preparation of need testing solution: take ginkgo-dipyridamine injection to be measured appropriate, the lyophilized powder amount containing dipyridamole 0.8mg Adding 20ml water dissolution, injection 20ml directly adds 2% hydrochloric acid solution 2, extracts 4 times with ethyl acetate shaking, each acetic acid second The consumption of ester is respectively 15,10,10,10ml, point take acetic acid ethyl fluid, with 5% sodium acetate solution 20ml washing, divide and take sodium acetate Liquid, then wash by ethyl acetate 10ml, divide and take ethyl acetate washings, wash with water 2 times, each 20ml, point water intaking washing liquid, use second Acetoacetic ester 10ml washs, combined ethyl acetate liquid, reclaims ethyl acetate the most dry, and residue adds methanol to be made dissolving and is transferred to 5ml amount In Ping, add methanol to scale, shake up, as need testing solution；

(2) preparation of reference substance solution: take ginkalide A, B, C and bilobalide appropriate, accurately weighed, add methanol and dissolve respectively Make every 1ml and contain 1mg ginkalide A, 1mg ginkalide B, 1mg ginkalide C, the solution of 2mg bilobalide, as comparison Product solution；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler；Mobile phase A is methanol, and Mobile phase B is Oxolane: 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, with evaporative light scattering detector detect, column temperature at 30 DEG C, The record time is 70min；Number of theoretical plate is calculated by bilobalide peak should be not less than 2500；

(4) formulation of standard finger-print: method described above is as formulating with Folium Ginkgo extract terpene lactone constituents feature It it is the means of testing of main finger printing；According to the collection of illustrative plates measured by 10 batches or more than 10 batches test samples, formulate standard fingerprint figure Spectrum, on the basis of the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, described In standard finger-print, total peak has 7；

The ratio having the total peak area of 30%～80% in ginkgo-dipyridamine injection finger printing the most to be measured refers to standard In stricture of vagina collection of illustrative plates, relatively, its difference cannot be greater than ± 30% to the odds ratio of each total peak area.

3. according to the detection method of the ginkgo-dipyridamine injection described in claim 1, it is characterised in that: assay include as Lower content:

(1) preparation of need testing solution: it is appropriate that precision weighs ginkgo-dipyridamine injection to be measured, injection 10ml volatilizes rear or freezes The dry powder amount containing dipyridamole 4mg directly puts methanol-25% hydrochloric acid solution mixed solution 25ml, is heated to reflux 30 minutes, lets cool, Filtration is transferred in 25ml measuring bottle, adds methanol to graduation mark, shakes up, as need testing solution；

(3) chromatographic condition: chromatographic column uses 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, detection Wavelength is 360nm, column temperature is 35 DEG C, and the record time is 90min, and number of theoretical plate is calculated by Quercetin peak should be not less than 2500；

(4) algoscopy: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, surveys Fixed；

(5) testing result: in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection or every bottle of ginkgo-dipyridamole for injection, quercetin content must not Less than 0.105mg, kaempferide content must not be less than 0.177mg, and isorhamnetin content must not be less than 0.048mg；

(3) chromatographic condition: chromatographic column uses octadecylsilane chemically bonded silica to be filler: mobile phase A is methanol, and Mobile phase B is Oxolane: 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, with evaporative light scattering detector detect, column temperature at 30 DEG C, record Time is 70min；Number of theoretical plate is calculated by bilobalide peak should be not less than 2500；

(4) algoscopy: precision draws reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid, surveys Fixed；

(5) testing result: in this product every 10ml Ginkgo Leaf Extract and Dipyridamole Injection or every bottle of ginkgo-dipyridamole for injection, ginkalide A content is not Must be less than 0.061mg, ginkalide B content must not be less than 0.022mg, and ginkalide C content must not be less than 0.040mg, Semen Ginkgo Lactone content must not be less than 0.087mg.

4. according to the detection method of the arbitrary described ginkgo-dipyridamine injection of claim 1-3, it is characterised in that: described note Penetrate preparation be directly used in drug administration by injection injection, need to dilute after for the concentrated solution for injection of intravenous drip, directly for quiet Glucose intravenous infusion that arteries and veins instils or sodium chloride intravenous infusion or the injection prepared with freeze-drying or spray drying method With sterilized powder or aseptic block.