CN111983077B

CN111983077B - Detection method of ginkgo dipyridamole injection preparation

Info

Publication number: CN111983077B
Application number: CN202010831588.0A
Authority: CN
Inventors: 窦啟玲; 杨青波; 陆煜玫; 段毅
Original assignee: Guizhou Yibai Pharmaceutical Co Ltd
Current assignee: Guizhou Yibai Pharmaceutical Co Ltd
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-08-30
Anticipated expiration: 2040-08-18
Also published as: CN111983077A

Abstract

The invention provides a detection method of a ginkgo dipyridamole injection preparation, wherein the ginkgo dipyridamole injection preparation is prepared from ginkgo biloba extract and dipyridamole, and the detection method comprises the items of properties, identification, inspection, fingerprint spectrum and content measurement. Compared with the prior art, the invention adds a thin-layer chromatography identification method for ginkgo leaf extract, limited inspection of 5-hydroxymethyl furfural, total ginkgoic acid and the like, inspection of related substances of injection, fingerprint detection and content determination methods for rutin, kaempferol-3-O-rutinoside, narcissus and terpene lactone, optimizes the content determination method for total flavonol glycoside, improves the requirement on product quality detection, is more suitable for quality control of future medicines and is more beneficial to ensuring the medicine quality and clinical efficacy.

Description

Detection method of ginkgo dipyridamole injection preparation

Technical Field

The invention belongs to the field of medicines, and particularly relates to a detection method of a ginkgo dipyridamole injection preparation.

Background

Cardiovascular and cerebrovascular diseases such as coronary heart disease, cerebral thrombosis, hypertension, cerebral infarction and the like are one of the most common and most harmful diseases in the world at present, are common diseases and frequently encountered diseases which are harmful to the health of people in China, and become one of the main causes of population death; it has been reported that the incidence of disease has been increasing year by year, and the number of middle-aged and young patients has been increasing. Ginkgo dipyridamole injection is prepared from ginkgo biloba extract and dipyridamole, and is loaded in national standards (first volume) of local standard upgrade of chemical drugs, and the standard number is as follows: WS-10001- (HD-0087) -2002. The product can resist platelet aggregation, belongs to coronary artery dilating drugs, is suitable for preventing and treating coronary heart disease and thromboembolic diseases, wherein the ginkgo leaf extract contains active ingredients such as flavonoids, terpenoids, phenols and the like, can play a role in improving myocardial and brain tissue metabolism, increasing coronary artery and cerebral vascular blood flow and improving blood supply of myocardial and brain tissues and the like, and has better curative effects on treating cardiovascular and cerebrovascular diseases such as coronary heart disease, angina pectoris, arrhythmia, cerebral thrombosis, senile dementia and the like.

In the early research, some detection indexes are gradually increased on the basis of quality standards, and meanwhile, the processes of reagents, detection means or sample preparation and the like in part of detection links are optimized. For example, the quality control method of CN200510200658.8 ginkgo dipyridamole injection preparation adds the thin layer identification of ginkgo biloba contrast extract and quercetin, kaempferide, isorhamnetin, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, and also adds the measuring method of terpene lactone content; CN 201310521033.6A method for detecting Ginkgo dipyridamole injection comprises fingerprint detection of folium Ginkgo extract and terpene lactone. However, the product is not brought into the national standard all the time, and with the development of society, the requirement on the quality control of the medicine is higher and higher, detection indexes such as identification, content measurement and the like of active ingredients in the medicine are required to be clear gradually, the requirement on searching for a quick, convenient and controllable detection method is urgent, technical requirements (temporary) on the research on the fingerprint of the traditional Chinese medicine injection are issued by the State food and drug administration, the traditional Chinese medicine fingerprint is expected to be loaded into the pharmacopoeia of the people's republic of China as a mandatory quality measure, and powerful quality guarantee is provided for the traditional Chinese medicine to move to the world. Therefore, under the dual requirements of external cause and internal cause, how to effectively control the quality of the product, ensure the clinical efficacy of the product, and meet the requirements of national standards on safety, effectiveness and the like is a problem which has been studied by the applicant for a long time.

Disclosure of Invention

In order to solve the problems and better control the product quality, the invention adds and revises the quality standard of the ginkgo dipyridamole injection on the basis of the original standard, and provides a detection method of the ginkgo dipyridamole injection preparation, which has good quality control effect and is specifically represented as follows: the limit inspection of harmful substances of 5-hydroxymethylfurfural and total ginkgoic acid is increased; fingerprint detection is added, fingerprint characterization is relatively comprehensive, the names of compounds to which 8 main common peaks in 15 common peaks belong are determined, the types and the amounts of internal chemical components of the product are objectively and truly reflected, the similarity is high compared with a comparison fingerprint, and the product quality of the ginkgo dipyridamole preparation can be effectively characterized; a thin layer identification method is added, and the method has strong specificity and good stability; the increased and optimized content measurement items are comprehensive, the method precision is high, the reproducibility is good, and the recovery rate is high; the detection method has accurate and reliable overall measurement result, improves the quality control standard of the ginkgo dipyridamole preparation, and can effectively control the product quality, thereby ensuring the clinical curative effect of the ginkgo dipyridamole preparation.

Specifically, the method comprises the following steps:

a detection method of a ginkgo dipyridamole injection preparation, wherein the ginkgo dipyridamole injection preparation is an injection prepared from ginkgo biloba extract and dipyridamole, and the detection method comprises part or all of characteristics, identification, inspection, fingerprint spectrum and content determination;

the method is characterized in that: the identification items comprise part or all of magnesium hydrochloride powder reaction identification for identifying flavonoid components, qualitative identification for dipyridamole chemical reaction, retention time identification for dipyridamole, total flavonol glycosides and terpene lactones, and thin layer identification for folium Ginkgo extract;

the inspection is partial or all of pH value inspection, solution color inspection, flavonoid aglycone peak area ratio inspection, burning residue inspection, total solid inspection, 5-hydroxymethylfurfural inspection, total ginkgoic acid inspection, heavy metal and harmful element residual amount inspection, protein inspection, tannin inspection, oxalate inspection, resin inspection, potassium ion inspection, pyrogen inspection and other injection project inspection;

the fingerprint is obtained by performing chromatographic characterization on main chemical components of folium Ginkgo extract and dipyridamole in the preparation by HPLC;

the assay comprises some or all of dipyridamole, total flavonol glycosides, rutin, kaempferol-3-O-rutinoside, narcissin, and terpene lactones content determination.

Further, the thin layer identification method of the ginkgo biloba extract comprises the following steps:

taking 20-100 ml of the product, passing through a macroporous adsorption resin column, eluting with 200-300 ml of water, discarding a water washing solution, eluting with 200-300 ml of ethanol, collecting an eluent, evaporating to dryness, adding 10-30 ml of n-butyl alcohol into residues, soaking in a water bath for 10-30 minutes while shaking, cooling, filtering, evaporating to dryness, adding 1-3 ml of ethanol into the residues to dissolve the residues to obtain a test solution; taking 0.1-0.5 g of ginkgo biloba leaf control extract, adding 10-30 ml of n-butyl alcohol, placing in a water bath, soaking for 10-30 minutes, shaking constantly, cooling, filtering, evaporating filtrate to dryness, and adding 1-3 ml of ethanol into residue to dissolve the residue to obtain a control extract solution; according to the test of Chinese pharmacopoeia thin-layer chromatography, 1-5 mul of each of the test sample solution and the control extract solution is respectively spotted on the same silica gel G thin-layer plate which takes sodium carboxymethyl cellulose solution containing 4% of sodium acetate as a binder, and the weight ratio of ethyl acetate: butanone: formic acid: 4-6: 2-4: 0.5-2: 0.5-2 of developing agent, developing, taking out, airing and spraying 3% -5% of alchlor ethanol solution; inspecting under 365nm ultraviolet lamp; the test sample shows fluorescent spots of the same color at the corresponding positions in the chromatogram of the control extract.

Furthermore, the thin-layer identification method of the ginkgo leaf extract comprises the following steps:

taking 50ml of the product, passing through a D101 type macroporous adsorption resin column, eluting with 250ml of water, discarding a water washing solution, eluting with 250ml of ethanol, collecting an eluent, evaporating to dryness, adding 15ml of n-butanol into residues, warm-soaking in a water bath for 15 minutes, shaking all the time, cooling, filtering, evaporating to dryness, adding 2ml of ethanol into the residues to dissolve the residues to obtain a sample solution; taking folium Ginkgo control extract 0.2g, adding n-butanol 15ml, placing in water bath, soaking for 15 minutes while shaking constantly, cooling, filtering, evaporating filtrate to dryness, and dissolving residue with ethanol 2ml to obtain control extract solution; according to the test of Chinese pharmacopoeia thin-layer chromatography, 1 microliter of each of the test solution and the control extract solution is respectively spotted on the same silica gel G thin-layer plate which takes sodium carboxymethyl cellulose solution containing 4% of sodium acetate as a binder, and the weight ratio of ethyl acetate: butanone: formic acid: water 5: 3: 1:1 is developing agent, taking out, airing, and spraying 3% aluminum trichloride ethanol solution; inspecting under 365nm ultraviolet lamp; the test sample shows fluorescent spots of the same color at the corresponding positions in the chromatogram of the control extract.

Further, the 5-hydroxymethylfurfural limit detection method comprises the following steps: according to the determination of the Chinese pharmacopoeia high performance liquid chromatography, octadecylsilane chemically bonded silica is used as a filling agent, and methanol or acetonitrile: 0.1-0.5% glacial acetic acid 2-20: 98-80 is a mobile phase, and the detection wavelength is 250-350 nm; taking a proper amount of a 5-hydroxymethylfurfural reference substance, precisely weighing, and adding methanol to prepare a reference substance solution; filtering the injection, and collecting the filtrate as test solution; precisely absorbing 5-10 mul of reference solution and sample solution respectively, injecting into a liquid chromatograph, and recording chromatogram; calculating the content of 5-hydroxymethylfurfural;

further, the 5-hydroxymethylfurfural inspection method comprises the following steps: the determination is carried out according to the high performance liquid chromatography of Chinese pharmacopoeia:

chromatographic conditions and system adaptability test by using octadecylsilane chemically bonded silica as a filler and methanol: 0.4% glacial acetic acid 4:96 is a mobile phase, the flow rate is 1.0ml per minute, the column temperature is 30 ℃, and the detection wavelength is 284 nm; the number of theoretical plates is not less than 4000 calculated according to the peak of 5-hydroxymethylfurfural;

preparation of reference solution an appropriate amount of 5-hydroxymethylfurfural reference solution was precisely weighed, and methanol was added to make 50 μ g/l solution as a reference solution;

preparing a test solution, namely taking the injection, filtering, and taking a subsequent filtrate as the test solution;

the determination method precisely absorbs 10 μ l of each of the reference solution and the sample solution, injects into the liquid chromatograph, and records the chromatogram; calculating the content of 5-hydroxymethylfurfural;

further, the total ginkgoic acid inspection method comprises the following steps: according to the determination of the high performance liquid chromatography in Chinese pharmacopoeia, octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile or methanol containing 0.1-0.5% of trifluoroacetic acid is used as a mobile phase A, water containing 0.1-0.5% of trifluoroacetic acid is used as a mobile phase B, gradient elution is carried out, and the detection wavelength is 250-350 nm; the gradient elution conditions were:

0-30min, mobile phase A75 → 90%, mobile phase B25 → 10%;

30-35min, mobile phase A90%, mobile phase B10%;

25-35min, mobile phase A17%, mobile phase B83%;

35-36min, mobile phase A90 → 75%, mobile phase B10 → 25%;

36-45min, mobile phase A75%, mobile phase B25%;

precisely weighing appropriate amount of neoacid reference substance, adding methanol to obtain reference solution, and preparing reference solution for positioning with methanol; filtering the injection to obtain a filtrate as a test solution; respectively and precisely sucking 10-50 μ l of each of the test solution, the reference solution and the positioning reference solution, injecting into a liquid chromatograph, and recording the chromatogram; calculating the total peak area of the corresponding chromatographic peak of the total ginkgoic acid reference substance in the test solution, and calculating the content of the total ginkgoic acid by using a ginkgolic neo-acid reference substance external standard method.

Furthermore, the total ginkgoic acid detection method comprises the following steps: the determination is carried out according to the high performance liquid chromatography of Chinese pharmacopoeia:

in chromatographic condition and system adaptability test, octadecylsilane chemically bonded silica is used as a filler, acetonitrile containing 0.1% of trifluoroacetic acid is used as a mobile phase A, water containing 0.1% of trifluoroacetic acid is used as a mobile phase B, gradient elution is carried out, and the detection wavelength is 310 nm. The number of theoretical plates is not lower than 4000 calculated according to the peak of neoacidity of ginkgo; the gradient elution conditions were:

0-30min, mobile phase A75 → 90%, mobile phase B25 → 10%;

30-35min, mobile phase A90%, mobile phase B10%;

25-35min, mobile phase A17%, mobile phase B83%;

35-36min, mobile phase A90 → 75%, mobile phase B10 → 25%;

36-45min, mobile phase A75%, mobile phase B25%;

preparation of reference solution A suitable amount of ginkgolic acid reference is precisely weighed, and methanol is added to make into 1 μ g solution per 1ml as reference solution. And preparing a proper amount of total ginkgoic acid reference substance into a solution containing 20 mu g of total ginkgoic acid per 1ml by using methanol, wherein the solution is used as a reference solution for positioning.

Preparing a test solution, namely taking the injection, filtering, and taking a subsequent filtrate as the test solution; the determination method precisely absorbs 50 μ l of each of the test solution, the reference solution and the positioning reference solution, injects into the liquid chromatograph, and records the chromatogram; calculating the total peak area of the corresponding chromatographic peak of the total ginkgoic acid reference substance in the test solution, and calculating the content of the total ginkgoic acid by using a ginkgolic neo-acid reference substance external standard method.

Further, the fingerprint detection method comprises the following steps:

the detection is carried out according to the high performance liquid chromatography of Chinese pharmacopoeia, and the specific method comprises the following steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; performing gradient elution by taking acetonitrile or methanol as a mobile phase A and taking a 0.1-0.5% phosphoric acid solution as a mobile phase B; the flow rate is 0.8-1.2 ml per minute; the detection wavelength is 200-400 nm; the gradient elution conditions were:

0-5min, mobile phase A8 → 12%, mobile phase B92 → 88%;

5-25min, mobile phase A12 → 17%, mobile phase B88 → 83%;

25-35min, mobile phase A17%, mobile phase B83%;

35-45min, mobile phase A17 → 23%, mobile phase B83 → 77%;

45-60min, mobile phase A23 → 25%, mobile phase B77 → 75%;

60-75min, mobile phase A25 → 60%, mobile phase B75 → 40%;

preparing reference solution by precisely weighing an appropriate amount of rutin reference substance, and dissolving in methanol to obtain reference solution;

preparing a test solution, filtering the product, and taking a subsequent filtrate to obtain the test solution;

the determination method precisely absorbs 5-10 mul of reference solution and test solution respectively, injects the solutions into a liquid chromatograph, determines, and records chromatogram map of 75 minutes, thus obtaining the test sample fingerprint map;

and (3) displaying chromatographic peaks with the same retention time as the chromatographic peaks of the reference substance in the fingerprint of the test sample, and calculating the similarity between the fingerprint of the test sample and the fingerprint of the reference substance according to a similarity evaluation system of the chromatographic fingerprints of the traditional Chinese medicine.

Furthermore, in the fingerprint detection method, in the chromatographic condition and system applicability test, the mobile phase A is acetonitrile, and the mobile phase B is 0.4% phosphoric acid solution; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 254 nm; the number of theoretical plates is not less than 10000 calculated according to rutin peak; the concentration of the reference substance solution is 0.1mg of rutin-containing methanol solution per 1 ml; the test sample fingerprint spectrum should have 15 common peaks, wherein the peak 4 is a pterosin peak, the peak 6 is a rutin peak, the peak 9 is a quercetin 3-O-glucosyl (1 → 2) rhamnoside peak, the peak 10 is a kaempferol-3-O-rutinoside peak, the peak 11 is a narcissus peak, the peak 13 is a 3-O- {2-O- [6-O- (p-hydroxy-trans-coumaroyl) -glucosyl ] -rhamnosyl } quercetin peak, the peak 14 is a 3-O- {2-O- [6-O- (p-hydroxy-trans-coumaroyl) -glucosyl ] -rhamnosyl } kaempferol peak, and the peak 15 is a dipyridamole peak; the common peak matching is calculated according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and the similarity between the fingerprint of the test sample and the fingerprint of the reference sample is not less than 0.90.

Further, the content determination method comprises the determination of dipyridamole, total flavonol glycosides, rutin, kaempferol-3-O-rutinoside, narcissus and terpene lactones, and the content determination method specifically comprises the following steps:

the method for measuring the dipyridamole content is measured according to the high performance liquid chromatography of Chinese pharmacopoeia, and comprises the following steps:

chromatographic conditions and system applicability test by using octadecylsilane chemically bonded silica as a filler, methanol or acetonitrile: 0.1-0.5% sodium dihydrogen phosphate solution with pH value of 3-6, 80-50: 20-50 is mobile phase; the detection wavelength is 250-300 nm;

preparing reference solution by accurately weighing appropriate amount of dipyridamole, and adding methanol solution to obtain reference solution;

preparing a test solution, precisely measuring 1-2 ml of injection, placing the injection in a 25-50 ml measuring flask, adding a methanol solution to dilute the injection to a scale, and shaking up to obtain the test solution;

measuring the reference solution and the sample solution respectively by 10-20 μ l by a measuring method, respectively injecting into a liquid chromatograph, recording a chromatogram, and calculating the dipyridamole content by peak area according to an external standard method;

the method for measuring the content of the total flavonol glycosides is to measure according to the high performance liquid chromatography of Chinese pharmacopoeia, and comprises the following specific steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; and (2) adding methanol or acetonitrile: 0.1-0.5% phosphoric acid solution 20-60: 80-40 is mobile phase; the detection wavelength is 300-400 nm;

preparing reference substance solution by precisely weighing appropriate amount of quercetin reference substance, kaempferide reference substance, and isorhamnetin reference substance, and adding methanol to obtain reference substance solution;

preparing a test solution, precisely measuring 5-10 ml of injection, adding methanol: 33% hydrochloric acid solution ═ 6: 1, placing the mixed solution on a water bath, heating and refluxing for 60-90 minutes, quickly cooling to room temperature, transferring to a 50ml measuring flask, diluting to a scale with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a test solution;

the determination method precisely absorbs 5-10 mul of each of the reference solution and the test solution, injects the solution into a liquid chromatograph, records a chromatogram, and calculates the total flavonol glycoside content according to the following formula:

total flavonol glycoside content = (quercetin content + kaempferide content + isorhamnetin content) × 2.51;

the content determination method of rutin, kaempferol-3-O-rutinoside and narcissus is determined according to high performance liquid chromatography of Chinese pharmacopoeia, and comprises the following specific steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; methanol or acetonitrile, 0.1-0.5% phosphoric acid solution 10-40: 90-60 are used as mobile phases; the detection wavelength is 300-400 nm;

preparing reference solution by precisely weighing appropriate amount of rutin reference, kaempferol-3-O-rutinoside reference, and narcissus reference, and dissolving in methanol solution to obtain reference solution;

respectively and precisely sucking 5-10 μ l of reference solution and test solution by determination method, injecting into a liquid chromatograph, recording chromatogram, and respectively calculating rutin, kaempferol-3-O-rutinoside and narcissus glycoside content;

the method for measuring the terpene lactone content is measured according to the high performance liquid chromatography of Chinese pharmacopoeia, and comprises the following steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; mixing the raw materials in a ratio of n-propanol: tetrahydrofuran (tetrahydrofuran): water is a mobile phase with the ratio of 1:10-20: 89-79; detecting with an evaporative light scattering detector;

preparing a reference extract solution, precisely weighing a proper amount of ginkgo leaf total lactone reference extract, and adding methanol to prepare a reference extract solution;

preparing a test solution, precisely measuring 15-25ml of injection, adding 2 drops of 2% hydrochloric acid solution, shaking and extracting with ethyl acetate for 5 times, 20-30 ml for the 1 st time, and 15-25ml for the rest 4 times, combining ethyl acetate extracting solutions, washing with 15-30 ml of 5% sodium acetate solution, collecting sodium acetate solution, and washing with 10-30 ml of ethyl acetate; combining the ethyl acetate extracting solution and the washing solution, washing for 2 times with water, 10-30 ml each time, separating the water solution, washing with 10-30 ml of ethyl acetate, combining the ethyl acetate solutions, recovering the solvent until the solvent is dry, dissolving the residue with methanol, transferring the residue into a 2-5 ml measuring flask, adding methanol to the scale, shaking up, filtering, and taking the subsequent filtrate to obtain the sample solution;

and precisely absorbing 3-5 mul and 10-20 mul of the reference extract solution and 5-10 mul of the test solution respectively by a determination method, injecting the solutions into a liquid chromatograph, recording a chromatogram, and respectively calculating the contents of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C by using an external standard two-point method logarithmic equation.

Furthermore, the content determination method comprises the following specific steps:

chromatographic conditions and system applicability test using octadecylsilane chemically bonded silica as filler, methanol: 0.1% sodium dihydrogen phosphate solution 75: 25 is a mobile phase, and the 0.1 percent sodium dihydrogen phosphate solution is adjusted to the pH value of 4.6 by a 1 → 3 phosphoric acid solution in advance; flow rate 1.0ml per minute; the detection wavelength is 290 nm; the number of theoretical plates is not less than 2000 calculated according to dipyridamole peak;

preparing a control solution by precisely weighing a proper amount of dipyridamole control, and adding 80% methanol solution to obtain a solution containing 15 μ g of dipyridamole per 1ml as the control solution;

preparing a test sample solution, precisely measuring 2ml of injection, placing the injection in a 50ml measuring flask, adding 80% methanol solution to dilute to a scale, and shaking up to obtain the test sample solution;

measuring the reference solution and the sample solution respectively by 20 μ l precisely, injecting into a liquid chromatograph, recording chromatogram, and calculating dipyridamole content according to external standard method and peak area;

the content determination method of the total flavonol glycosides is determined according to the high performance liquid chromatography of Chinese pharmacopoeia, and the specific method comprises the following steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; mixing methanol: 0.4% phosphoric acid solution ═ 50: 50 is a mobile phase; the detection wavelength is 360 nm; the theoretical plate number is not lower than 2500 calculated according to the peak of quercetin;

preparing reference solution by precisely weighing appropriate amount of quercetin reference, kaempferide reference and isorhamnetin reference, adding methanol to obtain mixed solution containing quercetin 0.03mg, kaempferide 0.025mg and isorhamnetin 0.01mg per 1ml, and shaking to obtain reference solution;

preparation of test solution 10ml of injection is precisely measured, and methanol is added: 33% hydrochloric acid solution ═ 6: 1, heating and refluxing the mixed solution for 60 minutes on a water bath, quickly cooling to room temperature, transferring to a 50ml measuring flask, diluting to a scale with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a test solution;

the determination method precisely absorbs 10 μ l of each of the reference solution and the sample solution, injects into a liquid chromatograph, records the chromatogram, and calculates the total flavonol glycoside content according to the following formula:

the content determination method of rutin, kaempferol-3-O-rutinoside and narcissus is determined according to high performance liquid chromatography of Chinese pharmacopoeia, and comprises the following steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile and 0.4 percent phosphoric acid solution are used as mobile phases, wherein the ratio of the acetonitrile to the 0.4 percent phosphoric acid solution is 16: 84; flow rate 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 360 nm; the theoretical plate number is not less than 5000 according to rutin peak;

preparing a reference solution by accurately weighing a proper amount of rutin reference, kaempferol-3-O-rutinoside reference and narcissus reference, adding 80% methanol to dissolve to obtain a mixed solution containing 0.08mg of rutin, 0.07mg of kaempferol-3-O-rutinoside and 0.06mg of narcissus per 1ml, and shaking up to obtain a reference solution;

preparing a test solution, namely filtering the injection and taking a subsequent filtrate to obtain the test solution;

measuring by accurately sucking 5 μ l of reference solution and test solution respectively, injecting into liquid chromatograph, recording chromatogram, and calculating rutin, kaempferol-3-O-rutinoside and narcissus glycoside content respectively;

the method for measuring the content of terpene lactone is measured according to the high performance liquid chromatography of Chinese pharmacopoeia, and comprises the following steps:

in chromatographic condition and system applicability test, octadecylsilane chemically bonded silica is used as a filler; mixing the raw materials in a ratio of n-propanol: tetrahydrofuran: water 1:15:84 is mobile phase; detecting with an evaporative light scattering detector; the number of theoretical plates is not less than 2500 calculated according to the peak of bilobalide;

preparing contrast extract solution by precisely weighing appropriate amount of folium Ginkgo total lactone contrast extract, and adding methanol to obtain 2.5mg solution per 1ml to obtain contrast extract solution;

preparing a test solution, precisely measuring 20ml of injection, adding 2 drops of 2% hydrochloric acid solution, shaking and extracting with ethyl acetate for 5 times, 20ml for the 1 st time, and 15ml for the other 4 times, combining ethyl acetate extract, washing with 20ml of 5% sodium acetate solution, separating sodium acetate solution, and washing with 10ml of ethyl acetate; mixing the ethyl acetate extractive solution and the washing solution, washing with water for 2 times (20 ml each time), separating water solution, washing with ethyl acetate 10ml, mixing ethyl acetate solutions, recovering solvent to dry, dissolving the residue with methanol, transferring to 2ml measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate to obtain sample solution;

and precisely absorbing 3 mu l and 10 mu l of the reference extract solution and 5-10 mu l of the test solution respectively by a determination method, injecting the reference extract solution and the test solution into a liquid chromatograph, recording a chromatogram, and respectively calculating the contents of bilobalide, bilobalide A, bilobalide B and bilobalide C by using an external standard two-point method logarithmic equation.

Specifically, a detection method of ginkgo dipyridamole injection preparation, which comprises the following steps:

the characteristics are as follows:

the injection is yellow to brown yellow clear liquid;

and (3) identification:

(1) taking 2ml of injection, adding a little magnesium powder, adding a few drops of concentrated hydrochloric acid, and standing to gradually show red;

(2) taking 2ml of injection, adding 10ml of ethanol to show green fluorescence, and adding diluted hydrochloric acid to remove the fluorescence;

(3) in chromatogram recorded under the items of measuring the contents of dipyridamole, total flavonol glycosides and terpene lactones, the retention time of the test sample peak is consistent with that of the corresponding reference sample peak and that of the ginkgo total lactone reference extract peak;

(4) taking 50ml of injection, passing through a D101 type macroporous adsorption resin column, eluting with 250ml of water, discarding a water washing solution, eluting with 250ml of ethanol, collecting an eluent, evaporating to dryness, adding 15ml of n-butanol into residues, warm-soaking in a water bath for 15 minutes, shaking constantly, cooling, filtering, evaporating to dryness, adding 2ml of ethanol into the residues to dissolve the residues to obtain a sample solution; collecting folium Ginkgo control extract 0.2g, adding n-butanol 15ml, and preparing control extract solution by the same method; according to the test of Chinese pharmacopoeia thin-layer chromatography, sucking 1 microlitre of the two solutions respectively, and respectively dropping the two solutions on the same silica gel G thin-layer plate which takes sodium carboxymethyl cellulose solution containing 4% of sodium acetate as a binder, and taking ethyl acetate: butanone: formic acid: water 5: 3: 1:1 is developing agent, taking out, airing, and spraying 3% aluminum trichloride ethanol solution; inspecting under 365nm ultraviolet lamp; fluorescent spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control extract;

and (4) checking:

(1) the pH value is checked according to a pH value measuring method of Chinese pharmacopoeia, and the pH value is 3.5-5.5;

(2) the peak area ratio of the flavone aglycone is calculated according to the chromatogram under the content measurement item of the total flavonol glycoside, the peak area ratio of the quercetin to the kaempferide is 0.8-1.2, and the peak area ratio of the isorhamnetin to the quercetin is more than 0.15;

(3) precisely measuring 10ml of injection for residues on ignition, evaporating to dryness, and checking according to residue on ignition check method in Chinese pharmacopoeia, wherein each 1ml of the residue on ignition should not exceed 1.0 mg;

(4) precisely measuring 10ml of total solid, placing the injection into an evaporation dish dried to constant weight, precisely adding 3g of diatomite dried to constant weight at 105 ℃ after evaporating in a water bath, stirring, placing the mixture into a dryer for drying for 3 hours at 105 ℃, moving the mixture into the dryer, cooling for 30 minutes, rapidly and precisely weighing the weight, deducting the added diatomite amount, and calculating that the total solid content in each 1ml of injection is 0.2-0.3 g;

(5) the 5-hydroxymethylfurfural is measured according to the high performance liquid chromatography of Chinese pharmacopoeia:

chromatographic conditions and system adaptability test by using octadecylsilane chemically bonded silica as a filler and methanol: 0.4% glacial acetic acid ═ 4:96 is a mobile phase, the flow rate is 1.0ml per minute, the column temperature is 30 ℃, and the detection wavelength is 284 nm; the number of theoretical plates is not less than 4000 calculated according to the peak of 5-hydroxymethylfurfural;

preparing a reference substance solution, precisely weighing a proper amount of 5-hydroxymethylfurfural reference substance, and adding methanol to prepare a solution containing 50 mu g of methanol per lml to serve as the reference substance solution;

(6) the total ginkgoic acid is measured according to the high performance liquid chromatography of Chinese pharmacopoeia:

0-30min, mobile phase A75 → 90%, mobile phase B25 → 10%;

30-35min, mobile phase A90%, and mobile phase B10%;

25-35min, mobile phase A17%, mobile phase B83%;

35-36min, mobile phase A90 → 75%, mobile phase B10 → 25%;

36-45min, mobile phase A75%, mobile phase B25%;

preparation of reference solution A suitable amount of neoacid ginkgolide reference was weighed precisely and added with methanol to make 1 μ g solution per 1ml as reference solution. Taking another appropriate amount of total ginkgolic acid reference substance, and preparing into solution containing 20 μ g per 1ml with methanol as reference solution for positioning;

preparing a test solution, namely filtering the injection and taking a subsequent filtrate as the test solution; the determination method precisely absorbs 50 μ l of each of the test solution, the reference solution and the positioning reference solution, injects into the liquid chromatograph, and records the chromatogram; calculating the total peak area of the corresponding chromatographic peak of the total ginkgoic acid reference substance in the test solution, and calculating the content of the total ginkgoic acid by using a ginkgolic neo-acid reference substance external standard method;

(7) the residual amounts of heavy metals and harmful elements are determined by the determination method of lead, cadmium, arsenic, mercury and copper in Chinese pharmacopoeia, each 1ml of injection contains lead of 0.24 mu g, cadmium of 0.06 mu g, arsenic of 0.12 mu g, mercury of 0.04 mu g and copper of 3.00 mu g;

(8) the protein is measured according to a Chinese pharmacopoeia injection related substance inspection method, 1ml of the product is taken, a newly prepared 30% sulfosalicylic acid solution lml is added, the mixture is uniformly mixed and placed for 5 minutes, no turbidity is generated, and if a component generating precipitation by acid exists in the injection, 1-3 drops of tannic acid test solution is added, no turbidity is generated;

(9) the tannin is determined according to Chinese pharmacopoeia injection related substance inspection method, and 1ml of the product is taken, and 5ml of newly prepared physiological sodium chloride solution containing 1% egg white is added (if necessary, the mixture is filtered by a microporous membrane (0.45 μm)), and the mixture is placed for 10 minutes without turbidity or precipitation. If turbidity or precipitation occurs, taking the injection lml, adding 1 drop of dilute acetic acid, and adding 4-5 drops of sodium chloride gelatin test solution to prevent turbidity or precipitation;

(10) the oxalate is determined according to a Chinese pharmacopoeia injection related substance inspection method, 5ml of the oxalate is taken, the pH value is adjusted to 1-2 by using dilute hydrochloric acid, the filtration is carried out, 2ml of filtrate is taken, the pH value of the filtrate is adjusted to 5-6, 2-3 drops of 3% calcium chloride solution are added, the mixture is placed for 10 minutes, and no turbidity or precipitation is caused;

(11) the resin is determined according to the inspection method of related substances of Chinese pharmacopoeia injection, 5ml of the product is taken, 1 drop of hydrochloric acid is added, and the product is placed for 30 minutes without precipitation. If precipitation occurs, adding 10ml of chloroform into 5ml of the product, shaking for extraction, separating chloroform solution, evaporating to dryness in water bath, dissolving the residue with 2ml of glacial acetic acid, placing in a test tube with a plug, adding 3ml of water, mixing, and standing for 30min to obtain no precipitation;

(12) measuring potassium ions according to a related substance inspection method of Chinese pharmacopoeia injection, taking 2ml of the product, evaporating to dryness, burning with small fire till charring, burning at 500-600 ℃ till completely incinerating, adding 2ml of dilute acetic acid for dissolving, placing into a 25ml measuring flask, adding water for diluting to a scale, and mixing uniformly to obtain a test solution. Taking two 10ml nano colorimetric tubes, precisely adding 0.8ml of standard potassium ion solution into the tube A, adding 0.6ml of alkaline formaldehyde solution (taking the formaldehyde solution, adjusting the pH value to 8.0-9.0 by using 0.1mol/L sodium hydroxide solution) and 2 drops of 3% ethylenediamine tetra-sodium acetate solution and 0.5ml of 3% tetraphenylboron sodium solution, adding water to dilute the formaldehyde solution into 10ml, precisely adding a sample solution lml into the tube B, carrying out simultaneous operation with the tube A, shaking up, arranging the tube A and the tube B on black paper, carrying out perspective from top to bottom, and comparing the turbidity displayed in the tube B with the tube A, wherein the turbidity cannot be more concentrated;

(13) diluting the pyrogen injection with 10% glucose injection by one time, examining according to Chinese pharmacopoeia pyrogen examination method, and slowly injecting 3ml of diluent with a dosage of 1 kg rabbit weight, wherein the dosage is in accordance with the regulation;

(14) other should accord with the relevant regulations under the Chinese pharmacopoeia injection item;

fingerprint spectrum: the determination is carried out according to the high performance liquid chromatography of Chinese pharmacopoeia:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; performing gradient elution by taking acetonitrile as a mobile phase A and 0.4% phosphoric acid solution as a mobile phase B; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 254 nm; the number of theoretical plates is not less than 10000 calculated according to rutin peak;

the gradient elution conditions were:

0-5min, mobile phase A8 → 12%, mobile phase B92 → 88%;

5-25min, mobile phase A12 → 17%, mobile phase B88 → 83%;

25-35min, mobile phase A17%, mobile phase B83%;

35-45min, mobile phase A17 → 23%, mobile phase B83 → 77%;

45-60min, mobile phase A23 → 25%, mobile phase B77 → 75%;

60-75min, mobile phase A25 → 60%, mobile phase B75 → 40%;

preparing reference solution by accurately weighing appropriate amount of rutin reference, and adding methanol to obtain 0.1mg solution per 1ml to obtain reference solution;

preparing a test solution, namely filtering the injection to obtain a subsequent filtrate to obtain the test solution;

the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, determining, and recording chromatogram for 75min to obtain sample fingerprint;

in the fingerprint of the test sample, a chromatographic peak with the same retention time as that of the chromatographic peak of the reference substance is presented, and the similarity of the fingerprint of the test sample and the reference fingerprint is calculated according to the similarity evaluation system of the chromatographic fingerprint of the traditional Chinese medicine;

content determination:

(1) dipyridamole is determined by high performance liquid chromatography according to Chinese pharmacopoeia:

chromatographic conditions and system applicability test using octadecylsilane chemically bonded silica as filler, methanol: 0.1% sodium dihydrogen phosphate solution 75: 25 is a mobile phase, and the 0.1 percent sodium dihydrogen phosphate solution is adjusted to the pH value of 4.6 by a 1 → 3 phosphoric acid solution in advance; the flow rate was 1.0ml per minute; the detection wavelength is 290 nm; the number of theoretical plates is not less than 2000 calculated according to dipyridamole peak;

preparing reference solution by accurately weighing appropriate amount of dipyridamole, and adding 80% methanol solution to obtain solution containing 15 μ g of dipyridamole per 1ml as reference solution;

preparing a sample solution, precisely weighing 2ml of injection, placing into a 50ml measuring flask, adding 80% methanol solution to dilute to scale, shaking, and collecting the filtrate to obtain the sample solution;

measuring 20 μ l of reference solution and sample solution by measuring accurately, respectively, injecting into liquid chromatograph, recording chromatogram, and calculating dipyridamole content according to external standard method and peak area;

(2) the total flavonol glycosides are determined by high performance liquid chromatography of Chinese pharmacopoeia:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; the method comprises the following steps of mixing methanol: 0.4% phosphoric acid solution ═ 50: 50 is a mobile phase; detecting the wavelength of 360 nm; the theoretical plate number is not lower than 2500 calculated according to the peak of quercetin;

preparing reference substance solution by accurately weighing appropriate amount of quercetin reference substance, kaempferide reference substance, and isorhamnetin reference substance, adding methanol to obtain mixed solution containing quercetin 0.03mg, kaempferide 0.025mg and isorhamnetin 0.01mg per 1ml, and shaking to obtain reference substance solution;

(3) the rutin, kaempferol-3-O-rutinoside and narcissus glycoside are measured by high performance liquid chromatography of Chinese pharmacopoeia:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.4% phosphoric acid solution (16: 84) is used as a mobile phase; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 360 nm; the theoretical plate number is not less than 5000 according to rutin peak;

preparation of reference solution A proper amount of rutin reference, kaempferol-3-O-rutinoside reference and narcissus reference is precisely weighed, 80% methanol is added to prepare a mixed solution containing 0.08mg of rutin, 0.07mg of kaempferol-3-O-rutinoside and 0.06mg of narcissus per 1ml, and the mixed solution is shaken up to obtain a reference solution;

(4) the terpene lactones are measured by high performance liquid chromatography according to Chinese pharmacopoeia:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; mixing the raw materials in a ratio of n-propanol: tetrahydrofuran: water 1:15:84 is mobile phase; detecting with an evaporative light scattering detector; the number of theoretical plates is not less than 2500 calculated according to the peak of bilobalide;

preparing a test solution by precisely measuring 20ml of injection, adding 2 drops of 2% hydrochloric acid solution, shaking and extracting with ethyl acetate for 5 times, 20ml for the 1 st time, and 15ml for the rest 4 times, mixing ethyl acetate extractive solutions, washing with 20ml of 5% sodium acetate solution, separating sodium acetate solution, and washing with 10ml of ethyl acetate; mixing the ethyl acetate extractive solution and the washing solution, washing with water for 2 times (20 ml each time), separating water solution, washing with ethyl acetate 10ml, mixing ethyl acetate solutions, recovering solvent to dry, dissolving the residue with methanol, transferring to 2ml measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate to obtain sample solution;

the determination method comprises precisely sucking 3 μ l and 10 μ l of reference extract solution and 5-10 μ l of test solution, respectively, injecting into liquid chromatograph, determining, and calculating bilobalide, bilobalide A, bilobalide B and bilobalide C content by external standard two-point method logarithmic equation.

Wherein, in the inspection items: the 5-hydroxymethylfurfural limit is as follows: each 1ml of injection contains not more than 0.01mg of 5-hydroxymethylfurfural; the total ginkgoic acid limit is as follows: each 1ml is filledThe injection contains no more than 15ng of total ginkgolic acid; in the fingerprint project: in the fingerprint of the test sample, there should be 15 common peaks, wherein the peak 4 is pterosin peak, the peak 6 is rutin peak, the peak 9 is quercetin 3-O-glucosyl (1 → 2) rhamnoside peak, the peak 10 is kaempferol-3-O-rutinoside peak, the peak 11 is narcissus peak, and the peak 13 is 3-O- {2-O- [6-O- (p-hydroxy-trans-coumaroyl) -glucosyl]A peak of-rhamnosyl } quercetin, a peak of No. 14 is 3-O- {2-O- [6-O- (p-hydroxy-trans-coumaroyl) -glucosyl]-rhamnosyl } kaempferol peak, peak No. 15 is dipyridamole peak; matching common peaks, and calculating according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint, wherein the similarity between the fingerprint of the test sample and the fingerprint of the reference sample is not lower than 0.90; in the content measurement items: dipyridamole (C) per 1ml injection ₂₄ H ₄₀ N ₈ O ₄ ) 0.36-0.44 mg; the total flavonol glycoside content of each 1ml injection is 0.85-1.10 mg; each 1ml of injection contains rutin (C) ₂₇ H ₃₀ O ₁₆ ) 0.06-0.12 mg of kaempferol-3-O-rutinoside (C) ₂₇ H ₃₀ O ₁₅ ) 0.05mg to 0.10mg of hydrastin (C) ₂₈ H ₃₂ O ₁₆ ) 0.04 mg-0.08 mg; the injection contains terpene lactone and bilobalide (C) per 1ml ₁₅ H ₁₈ O ₈ ) Ginkgolide A (C) ₂₀ H ₂₄ O ₉ ) Ginkgolide B (C) ₂₀ H ₂₄ O ₁₀ ) And ginkgolide C (C) ₂₀ H ₂₄ O ₁₁ ) The total amount should not be less than 0.18 mg.

The invention has the beneficial effects that:

1. a thin layer identification method of the ginkgo leaf extract is newly added, a development system, a test sample preparation method, a color developing agent, a sample amount and the like are selected and optimized, and the problems of spot trailing, poor specificity and the like are avoided. Compared with the quality control method of CN200510200658.8 ginkgo dipyridamole injection, the method has the advantages of simple treatment method of the sample, less toxicity of the developing agent, clearer spot, better separation effect, stronger specificity, and more suitability for effectively controlling the ginkgo leaf extract in the product.

2. The method increases the peak area ratio of the flavonoid aglycone, the detection of burning residues and total solids, increases the limit detection of 5-hydroxymethylfurfural and total ginkgoic acid, changes the heavy metal detection of the original colorimetric method into the detection of the residual quantity of heavy metals and harmful elements determined by adopting an atomic absorption spectrophotometry, and can effectively control the product quality.

3. The inspection of related items of related substances of the injection, such as protein, tannin, oxalate, resin, potassium ions and the like, is increased, the product quality can be effectively controlled, and the occurrence of adverse reactions is reduced.

4. Compared with a detection method of CN201310521033.6 a ginkgo dipyridamole injection preparation, the method optimizes chromatographic conditions, effectively separates 15 common peaks, defines the compound names of 8 main common peaks in the 15 common peaks, and further shows that the process of the ginkgo dipyridamole injection preparation is stable and the product quality is controllable through the fingerprint detection of a plurality of batches of samples.

5. The method for measuring the contents of rutin, kaempferol-3-O-rutinoside and narcissus glycoside by an HPLC method is added, the sample treatment method and chromatographic conditions of the total flavonol glycoside are optimized, and the product quality is more controllable.

6. The method for measuring the terpene lactone content by an HPLC-ELSD method is added, the sample processing method and the chromatographic conditions of the terpene lactone are optimized, and the product quality is more controllable.

In conclusion, the detection method of the invention has accurate and reliable overall measurement result, improves the quality control standard of the ginkgo dipyridamole injection preparation, and can effectively control the product quality, thereby ensuring the clinical curative effect.

To better illustrate the advantageous effects of the invention, the following research procedures are also included in the present invention, which are intended to illustrate the advantageous effects of the invention, but are in no way limited to the scope of protection of the invention.

First, thin layer chromatography identification research of ginkgo leaf extract

In the prior art, the method for identifying ginkgo biloba extract is disclosed in the section of the 'Chinese pharmacopoeia' 2015 edition: taking a sample, adding n-butanol, extracting in warm water bath, using folium Ginkgo extract as control, using silica gel G thin layer plate containing 4% sodium acetate sodium carboxymethylcellulose solution as binder, using ethyl acetate-butanone-formic acid-water (5: 3: 1) as developing agent, using 3% aluminum trichloride ethanol solution as color developing agent, and inspecting under ultraviolet light (365 nm).

The present invention also refers to this thin layer chromatography for differential studies.

Research on preparation method of test solution

The invention firstly selects the sample to be sampled after being extracted by the n-butyl alcohol, and then the sample is spotted, so that the sample solution is thicker, the spotting plate is not good, and the sample developing effect is not good, and then the sample is purified by a column and then extracted by the n-butyl alcohol, so the effect is better, therefore, the preparation method of the sample is determined as follows:

taking 50ml of the product, passing through a D101 type macroporous adsorption resin column (with an inner diameter of 2cm and a column height of 27cm), eluting with 250ml of water, discarding water washing solution, eluting with 250ml of ethanol, collecting eluent, evaporating to dryness, adding 15ml of n-butanol into residue, soaking in a water bath for 15 minutes while shaking, cooling, filtering, evaporating filtrate to dryness, and adding 2ml of ethanol into residue to dissolve to obtain a sample solution.

Study of specificity tests

Firstly, preparing a negative sample solution, wherein the specific method comprises the following steps: taking appropriate amount of adjuvants (except folium Ginkgo extract and dipyridamole) for preparing YINXINGDAMO injection, making into aqueous solution according to YINXINGDAMO injection preparation process, and making into negative control solution according to test solution preparation method; then respectively taking 1ul of the test solution, the control extract solution and the negative control solution, respectively dropping on the same silica gel G thin-layer plate which takes sodium carboxymethylcellulose solution containing 4% of sodium acetate as an adhesive, taking ethyl acetate-butanone-formic acid-water (5: 3: 1) as a developing agent, developing, taking out, airing, and spraying with 3% aluminum trichloride ethanol solution. Inspecting under ultraviolet light (365 nm). The result shows that the test sample shows fluorescence spots with the same color at the corresponding positions of the chromatogram of the control extract. Negative controls had no effect on the identification. The authentication specificity is good.

Examination of spotting method and spotting amount

The sample solution to be tested and the reference solution are subjected to point sample application and strip sample application, and the result shows that the strip sample application is clearer.

By inspecting the different point sample amount of 0.5-5 mu l (strip shape),

the result shows that the thin layer effect is best when the sample application amount is 1-3 mu l.

Durability examination

The examination of the development effect of different development temperatures (7 ℃, 40 ℃ and normal temperature) and humidities (75%, 45% and 20% of humidity) shows that the difference of the development effect of different temperatures and humidities is not large, so that the temperature and humidity changes have no influence on the identification.

Results of the study

The thin-layer identification method of the ginkgo leaf extract is obtained by combining the following research results: taking 50ml of the product, passing through a D101 type macroporous adsorption resin column (with an inner diameter of 2cm and a column height of 27cm), eluting with 250ml of water, discarding water washing solution, eluting with 250ml of ethanol, collecting eluent, evaporating to dryness, adding 15ml of n-butanol into residue, soaking in a water bath for 15 minutes while shaking, cooling, filtering, evaporating filtrate to dryness, and adding 2ml of ethanol into residue to dissolve to obtain a sample solution. Taking 0.2G of ginkgo dipyridamole contrast extract, adding 15ml of n-butanol, preparing contrast extract solution by the same method, respectively taking 1ul of test sample solution and contrast extract solution, respectively dropping on a silica gel G thin layer plate which uses sodium carboxymethylcellulose solution containing 4% of sodium acetate as adhesive, developing by using ethyl acetate-butanone-formic acid-water (5: 3: 1) as developing agent, taking out, drying in the air, and spraying with 3% aluminum trichloride ethanol solution. Inspecting under ultraviolet light (365 nm).

Thin layer identification was performed by applying this method to 30 batches of samples. The results show that the main bands of the sample are basically consistent compared with the spectra of the control extract, which indicates that the method has better durability and can identify the specificity of the ginkgo dipyridamole injection.

Limit examination research of di, 5-hydroxymethyl furfural and total ginkgoic acid

1. 5-hydroxymethylfurfural assay

5-hydroxymethyl furfural (5-HMF) is an aldehyde compound generated by monosaccharide compounds such as glucose under the conditions of high temperature or weak acid, and the literature reports that the 5-HMF has side effects on a human body and certain damage to striated muscles and internal organs of the human body. In recent years, the Chinese patent medicine pays more and more attention to the determination and control of 5-HMF, but the original standard of the injection does not carry out limit control on the 5-HMF, and in order to establish a more perfect quality control method of the ginkgo dipyridamole injection and ensure the safety of the medicine, a limit inspection method of the 5-hydroxymethylfurfural in the ginkgo dipyridamole injection is established through research.

The main research points are as follows: reference is made to the literature "preliminary discussion of determining the content of 5-hydroxymethylfurfural in shenqi strengthening injection by HPLC method and the origin thereof" (liujiagjie et al. journal of drug analysis, 2012,32 (4)) and "high performance liquid chromatography determination of the content of 5-hydroxymethylfurfural in shenmai injection (shangmei et al, shi zhen national medicine, 2010,21 (7)) regarding the method for determining the content of 5-hydroxymethyl in other varieties of Chinese medicine injections, wherein the RP-HPLC-UV method is used to search the chromatographic conditions for determining the component in the ginkgo-dipyridamole injection, and the mobile phase compositions such as acetonitrile-water, acetonitrile-acid water, methanol-acid water and the like are respectively examined, as a result, the product has a large polarity, and an acetonitrile-water system is used, no suitable method is found, and the methanol-water system and the different methanol-acid water systems are compared, as a result, the peak profile and resolution of the chromatographic peak of the 5-HMF component in the injection were optimized under the condition of methanol-0.4% glacial acetic acid (4: 96), and the mobile phase composition was finally determined to be methanol-0.4% glacial acetic acid (4: 96).

Further investigation and research on flow rates of a chromatographic column and a column temperature column, a preparation method of a test sample, precision of an instrument, repeatability, accuracy and other methodologies are carried out, 30 batches of ginkgo dipyridamole injection samples are used for verifying the obtained examination conditions (see example 1), the result shows that the method is sensitive and effective, the content range of the 5-hydroxymethylfurfural is 0.2-2.6 mu g/ml, the specification of the limit of the 5-hydroxymethylfurfural in the glucose injection and the glucose sodium chloride injection of the second part of the version of Chinese pharmacopoeia 2015 is used for reference, and the usage amount of the ginkgo dipyridamole injection is combined, so that the content of the 5-hydroxymethylfurfural in each 1ml of the product is temporarily determined to be not more than 0.01 mg. Therefore, the inspection result of the method is far lower than the requirement specified by pharmacopoeia (equivalent conversion).

2. Total ginkgolic acid assay

The total ginkgoic acid is a check item in the quality standard of the ginkgo biloba extract, but the quality standard of the ginkgo dipyridamole injection is not detected all the time, in order to effectively control the product quality, the applicant refers to the chromatographic conditions of the total ginkgoic acid in the quality standard of the ginkgo biloba extract to verify the ginkgo biloba extract, and the separation degree, the sensitivity and the like of the result meet the requirements, so the method is also suitable for the ginkgo dipyridamole injection. The total ginkgolic acid of 30 batches of ginkgo dipyridamole injection samples is determined by the method, and the result is not detected. According to the content limit of total ginkgolic acid in ginkgo biloba extract of 2020 edition of Chinese pharmacopoeia, the content of the total ginkgolic acid in each 1ml of the product is temporarily determined to be not more than 15 ng.

Third, research of fingerprint

Although ginkgo-dipyridamole fingerprint detection methods are disclosed in patent document 201310521033.6, the present applicant has conducted further studies on ginkgo-dipyridamole injection fingerprints because the degree of separation between peaks is not good and the identification of the components of the common peaks is low.

Chromatographic conditions and System suitability test

1.1. Investigation of detection wavelength

6 detection wavelengths were investigated: the chromatographic peaks at 203nm, 210nm, 230nm, 254nm, 280nm and 360nm are better reflected under 254nm, and the base line is stable, so 254nm is selected as the detection wavelength of the fingerprint of the ginkgo-dipyridamole injection.

Investigation of chromatographic columns

3 types of chromatographic columns, namely an Agilent ZORBAX SB-C18 chromatographic column, an Aglea Durashell C18 chromatographic column and a Kromasil 100-5-C18 chromatographic column, are selected for study, and as a result, the Kromasil 100-5-C18 chromatographic column has good separation effect and good peak shape, so the Kromasil 100-5-C18 chromatographic column is selected.

Investigation of mobile phase

3 mobile phase systems were selected: the acetonitrile-water gradient system, the acetonitrile-0.4% phosphoric acid water gradient system and the acetonitrile-0.5% formic acid water gradient system are adopted, and the acetonitrile-0.4% phosphoric acid water gradient system is adopted as a good result, the separation degree of each chromatographic peak is good, and the retention time is moderate, so the acetonitrile-0.4% phosphoric acid water gradient system is selected as a mobile phase.

Investigation of column temperature

Column temperatures were investigated separately: 25 deg.C, 30 deg.C, 35 deg.C. As a result, the separation degree of each chromatographic peak was good at a column temperature of 30 ℃.

Investigation of flow velocity

The flow rates were investigated separately: 0.8ml/min, 1.0ml/min, 1.2 ml/min. As a result, the resolution of each chromatographic peak was good at a flow rate of 1.0 ml/min.

Methodology survey

2.1. Investigation of solvents and time

According to the preliminarily selected chromatographic conditions, chromatographic analysis is respectively carried out on the mobile phase, the blank solvent and various auxiliary materials, the result has no chromatographic peak, and the chromatographic analysis with 2 times retention time is carried out on the ginkgo dipyridamole injection. The result shows that the test article has no other chromatographic peak after 75 min.

Precision test

And (3) sampling, preparing a sample according to a preparation method of the text sample solution, continuously sampling for 6 times, and detecting the fingerprint, wherein the result shows that the similarity of each spectrum peak meets the requirement of the fingerprint.

Repeatability test

And (3) sampling, preparing 6 parts of a test sample according to the preparation method of the text test sample solution, and detecting a fingerprint, wherein the result shows that the relative retention time of each chromatographic peak and the similarity of the chromatographic peaks both accord with the requirement of the fingerprint.

Stability test

A sample is prepared according to the preparation method of the text test sample solution, and the fingerprint spectrums are detected in 0, 3, 6, 9, 12 and 40 hours respectively, and the result shows that the similarity of each spectrum peak meets the requirement of the fingerprint spectrum.

Sample assay

By performing fingerprint analysis on 30 batches of ginkgo dipyridamole injection, the similarity of chromatographic peaks of all batches of samples meets the requirement of the fingerprint.

Selection of reference fingerprint

The fingerprint detection results of 30 ginkgo dipyridamole injection test samples are analyzed and compared, and the fingerprint of one test sample is determined to be used as a reference fingerprint, because the relative retention time of the test sample is closer to the average relative retention time of each test sample, and the separation degree is better, so the test sample is a more typical fingerprint.

Common fingerprint peak calibration

The detection results of fingerprint patterns of 30 batches of ginkgo dipyridamole injection samples are very similar no matter from the number of peaks, the area of the peaks and the retention time, typical chromatographic peaks appearing in the fingerprint patterns of each batch of the samples are taken as common fingerprint peaks, 15 common peaks are totally obtained, wherein the peak No. 6 is the chromatographic peak of rutin and is represented by 'S', and the chromatographic peaks are represented by serial numbers of 1-15 respectively. Peak number of common peaks (relative retention time): 1 (0.24), 2 (0.32), 3 (0.72), 4 (0.89), 5 (0.91), 6 (S peak) (1.00), 7 (1.06), 8 (1.09), 9 (1.24), 10 (1.27), 11 (1.35), 12 (1.64), 13 (1.84), 14 (2.01), 15 (2.31). Peak shape description of part of the common peaks: peaks No. 1-15 basically reach baseline separation, and except peaks No. 1, 2, 5, 7 and 8, the peaks are higher.

Establishment of fingerprint spectrum similarity detection standard of ginkgo dipyridamole injection

The detection results of 30 batches of ginkgo dipyridamole injection test sample fingerprints are analyzed by adopting software of 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system, 2012 edition' to generate a comparison map, and the result shows that the similarity values of the fingerprint of the test sample are all more than 0.995 compared with the common mode, so the text specifies: the sample fingerprint and the reference fingerprint should have the same chromatographic peak at corresponding retention time, and the similarity between the two should not be less than 0.90.

Fingerprint spectrum correlation calibration of folium Ginkgo medicinal material, folium Ginkgo extract, and YINXINGDAMO injection

The results of the researches on the fingerprint spectrums of the ginkgo dipyridamole injection and the researches on the fingerprint spectrums of the ginkgo biloba leaves and the ginkgo biloba extracts show that the ginkgo dipyridamole injection contains the main information of the ginkgo biloba leaves and the ginkgo biloba extracts and has good correlation.

Description of the relevant peaks: except the peak No. 1 in the ginkgo dipyridamole injection, the peaks No. 2 to 14 can be corresponded to the ginkgo biloba leaves and the ginkgo biloba extract.

Fourthly, content determination

1. Research on total flavonol glycoside content determination method

Based on the original quality standard, the sample processing method is optimized, the sample processing time is saved, and meanwhile, the quercetin, kaempferide and isorhamnetin are used as the reference, so that the content calculation of the total flavonol glycoside is more accurate.

1.1. Study on sample treatment method

1.1.1. Investigation of Water bath reflux extraction time

By comparing the water bath heating reflux extraction time (30 minutes, 60 minutes and 90 minutes), the result shows that the total flavonol glycosides can be completely hydrolyzed after the water bath reflux is carried out for 60 minutes, and the result of the water bath reflux for 90 minutes is equivalent to the result of 60 minutes, so the water bath reflux extraction is selected for 1 hour. See Table 1 for details

TABLE 1 reflux extraction time survey

Time of reflux	30min	60min	90min
				Content (mg/ml)	0.9219	0.9370	0.9505

1.1.2. Investigation of amount of extraction solvent

The results of the comparative extraction investigation by adding different amounts (15 ml, 25ml and 35 ml) of mixed solution of methanol-33% hydrochloric acid (4: 1) show that the extraction effect of total flavonol glycosides is best when 35ml of solvent is used, so 35ml of solvent is used for extraction and reflux. See table 2 for details:

TABLE 2 examination of the amount of solvent extracted

Amount of extraction solvent	15ml	25ml	35ml
				Content (mg/ml)	0.8567	0.9469	0.9789

1.1.3. Examination of the ratio of methanol to 33% hydrochloric acid

By adding methanol-33% hydrochloric acid (4: 1) and methanol-33% hydrochloric acid (6: 1) for extraction by comparison, the results show that the content of total flavonol glycosides is unstable when methanol-33% hydrochloric acid (4: 1) is added, the repeatability of the method is poor, and therefore, the proportion of methanol-33% hydrochloric acid is selected from 6: 1. see table 3 for details:

TABLE 3 examination of the ratio of methanol to 33% hydrochloric acid

1.1.4. Examination of hydrochloric acid concentration

The results of the extraction by adding the mixed solution of methanol-30% hydrochloric acid (6: 1), methanol-33% hydrochloric acid (6: 1) and methanol-36% hydrochloric acid (6: 1) show that the total flavonol glycosides can be extracted completely by using 33% hydrochloric acid, the results of the 33% hydrochloric acid and the 36% hydrochloric acid are equivalent, the RSD% is 0.68%, and therefore the hydrochloric acid concentration is selected to be 33%. See table 4 for details:

TABLE 4 hydrochloric acid concentration investigation

Amount of extraction solvent	30% hydrochloric acid	33% hydrochloric acid	36% hydrochloric acid
				Content (mg/ml)	0.9338	0.9601	0.9509

1.2. Methodology investigation

1.2.1 precision investigation

A sample is taken and prepared according to the preparation method of the text test sample solution, the sample is continuously injected for 6 times, and a chromatogram is recorded, and the result shows that the precision of the instrument is good.

1.2.2. Repeatability test

Taking a sample, preparing 6 parts of test sample according to the text test sample solution preparation method, injecting the test sample into a liquid chromatograph, and recording a chromatogram, wherein the average content of the total flavonol glycosides is 0.9604mg/ml, and the RSD is 0.91 percent, which shows that the method has good repeatability.

1.2.3. Stability test

Taking a sample, preparing a test sample solution according to the preparation method of the text test sample solution, and respectively measuring the peak areas of quercetin, kaempferide and isorhamnetin within 0,2, 4, 6, 12 and 24 hours, wherein the result shows that the test sample solution has good stability within 24 hours.

1.2.4. Test of recovery

By adopting a sample-adding recovery method, the average recovery rate of the quercetin is 95.81 percent, and the RSD is 3.86 percent; the average recovery rate of the kaempferide is 95.56 percent, and the RSD is 2.45 percent; the average recovery rate of isorhamnetin is 95.53%, and the RSD is 4.28%. The results show that the process yields good.

1.2.5. Durability test

Samples were prepared as described for the test solutions and measured on different columns [ Agela Durashell C18 (4.6X 250mm, 5 μm), GRACE Prevail C18 (4.6X 250mm, 5 μm), Agilent ZORBAX SB-C18 (4.6X 250mm, 5 μm), Agilent ZORBAX extended-C18 (4.6X 250mm, 5 μm), CAPCELL PAK C18 BB (4.6X 250mm, 5 μm) ], different flow rates (0.8, 1.0, 1.2 ml/min), different column temperatures (20, 25, 30, 35, 40 ℃). The result shows that the determination of different chromatographic columns, different flow rates and different column temperatures only changes the peak output time, and has no influence on the determination result of the total flavonol glycoside content, which indicates that the method has good durability.

1.3. Sample assay

The content of the total flavonol glycosides in 30 batches of ginkgo dipyridamole injection is determined to be 0.88-0.98 mg/ml, and the average value is 0.95mg/ml, so that the total flavonol glycosides in the injection is determined to be 0.85-1.10 mg/ml.

Research on methodology of rutin, kaempferol-3-O-rutinoside and narcissus

In the prior art, rutin, kaempferol-3-O-rutinoside and narcissin are not determined in the ginkgo dipyridamole injection, and the 3 components are different from quercetin, kaempferide and isorhamnetin, so that the determination is carried out to better control the product quality.

2.1. System suitability test

And (3) respectively injecting rutin, kaempferol-3-O-rutinoside and narcissus glycoside mixed reference substance solution, test sample solution and negative test sample solution lacking folium Ginkgo extract into a liquid chromatograph, and recording chromatogram. From the chromatogram, the peak emergence times of rutin, kaempferol-3-O-rutinoside and narcissus reference substances under the test condition are respectively about 15 minutes, 27 minutes and 32 minutes, the rutin and the kaempferol-3-O-rutinoside are completely separated from similar impurity peaks (the separation degree is more than 1.5), the narcissus peak is completely separated from the similar impurity peaks (the separation degree is more than 1.4), and negative samples have no interference.

2.2. Methodology survey

2.2.1. Investigation of linear relationships

Respectively taking a rutin control, a kaempferol-3-O-rutinoside control and a narcissus control, preparing and diluting the rutin control, determining peak areas, and performing linear regression calculation on the sample amount (mu g) by using the peak area A to obtain a rutin linear range: 0.0818 mug-2.0449 mug, linear range of kaempferol-3-O-rutinoside: 0.0733-1.8323 mug; linear range of narcissus: 0.0665-1.6628 μ g.

2.2.2. Precision survey

Sampling, preparing a sample according to a preparation method of a text sample solution, continuously sampling for 6 times, and recording a chromatogram, wherein the peak area RSD of rutin is 0.34 percent; the peak area RSD of the kaempferol-3-O-rutinoside is 0.28 percent; the peak area RSD of the narcissus glycoside is 0.71%, which indicates that the precision of the instrument is good.

2.2.3. Repeatability test

Sampling, preparing 6 parts of test sample according to the preparation method of the test sample solution, injecting into a liquid chromatograph, and recording a chromatogram, wherein the average rutin content is 0.0864mg/ml, and the RSD is 0.86%; the average content of kaempferol-3-O-rutinoside is 0.0800mg/ml, and the RSD is 1.85 percent; the average narcissus glycoside content is 0.0691mg/ml, and RSD is 2.09%, which shows that the method has good repeatability.

2.2.4. Stability test

Taking a sample, preparing a test solution according to the text test solution preparation method, and measuring the peak areas of rutin, kaempferol-3-O-rutinoside and narcissus glycoside at 0,2, 4, 6, 12 and 24 hours respectively, wherein the result shows that the test solution has good stability within 24 hours.

2.2.5. Recovery test

By adopting a sample-adding recovery method, the average recovery rate of rutin is 96.78 percent, and the RSD is 1.27 percent; the average recovery rate of the kaempferol-3-O-rutinoside is 97.42 percent, and the RSD is 1.17 percent; the average recovery rate of the narcissus glycoside is 99.66 percent, the RSD is 0.70 percent, and the result shows that the recovery rate of the method is good.

2.2.6. Durability test

Samples were prepared as described for the test solutions and tested on different columns [ Agela Durashell C18 (4.6X 250mm, 5 μm), GRACE Prevail C18 (4.6X 250mm, 5 μm), Agilent ZORBAX SB-C18 (4.6X 250mm, 5 μm), Agilent ZORBAX extended-C18 (4.6X 250mm, 5 μm), CAPCELL PAK C18 BB (4.6X 250mm, 5 μm) ], different flow rates (0.8, 0.9, 1.0, 1.2 ml/min), different column temperatures (20, 25, 30, 35, 40 ℃). The result shows that the determination of different chromatographic columns, different flow rates and different column temperatures only changes the peak-appearing time, has no influence on the determination results of the contents of rutin, kaempferol-3-O-rutinoside and narcissus, and shows that the method has good durability.

2.2.7. Sample assay

Through content determination of 30 batches of ginkgo dipyridamole injection, the determination result of rutin content is 0.076-0.093 mg/ml, the average value is 0.083 mg/ml, the determination result of kaempferol-3-O-rutinoside content is 0.050-0.079 mg/ml, and the average value is 0.066 mg/ml; the result of the narcissus content measurement is 0.040-0.071 mg/ml, and the average value is 0.055 mg/ml. Therefore, the proposed product contains 0.06-0.12 mg of rutin, 0.05-0.10 mg of kaempferol-3-O-rutinoside and 0.04-0.08 mg of narcissus in each 1 ml.

Terpene lactones methodology study

The content measurement research of terpene lactones contained in the product is carried out according to the terpene lactone content measurement method in the ginkgo biloba extract quality standard in Chinese pharmacopoeia.

Preparation of test solution

3.1.1. Examination of sample amount

According to the extraction method of folium Ginkgo extract in "Chinese pharmacopoeia" 2015 edition, samples (20 ml, 30ml and 40 ml) with different amounts are respectively prepared to be tested, and terpene lactone content is determined. The results showed that the sample amount was 20ml, which was higher in content and more complete in extraction, and thus the sample amount was determined to be 20 ml. See table 5 for details.

TABLE 5 examination of sample amounts

Sample size (ml)	Content (mg/ml)
		20	0.2627
30	0.2507
		40	0.2212

3.1.2. Investigation of sample size

Precisely sucking 5. mu.l, 6. mu.l, 7. mu.l, 8. mu.l, 9. mu.l and 10. mu.l of the same sample solution, respectively, injecting into a liquid chromatograph, and recording chromatogram. The result shows that the content is higher when the sample injection amount is 7-10 mu l.

Test of system suitability

3.2.1. Inspection of chromatographic columns

4 different chromatographic columns were used: agela Durashell C18 (4.6 × 250mm, 5 μm), GRACE Prevail C18 (4.6 × 250mm, 5 μm), Agilent ZORBAX SB-C18 (4.6 × 250mm, 5 μm), and Agilent ZORBAX extended-C18 (4.6 × 250mm, 5 μm) were examined for the separation effect of ginkgolide C, bilobalide, ginkgolide A, and ginkgolide B. The results show that the GRACE Prevail C18 chromatographic column has lower content of terpene lactones than those separated from other three columns; although the content of terpene lactones obtained by separating an Agelent Durashell C18 chromatographic column and an Agilent ZORBAX SB-C18 chromatographic column is highest, the collection time of the Agilent ZORBAX SB-C18 chromatographic column is longer, and the separation degree of the Agelent Durashell C18 chromatographic column is poorer than that of the Agilent ZORBAX extended-C18 chromatographic column, so that the Agilent ZORBAX extended-C18 chromatographic column is selected for subsequent tests.

Temperature investigation of drift tube

The temperatures of the three drift tubes were used: the separation effect and the content of the terpene lactones are examined at the temperature of 95 ℃, 100 ℃ and 105 ℃, and the result shows that when the temperature of the drift tube is 105 ℃, the terpene lactones content is high, the retention time is proper, so that the temperature of the drift tube is selected to be 105 ℃.

Investigation of air flow

Three different air flows were used: 2.5L/min, 3.0L/min and 3.5L/min, and the separation effect and the content of the terpene lactones are examined. The results show that the separation degree of terpene lactones is not greatly influenced by three different air flow rates, and when the air flow rate is 3.0L/min, the terpene lactone content is higher, so that the air flow rate is selected to be 3.0L/min.

Investigation of flow Rate

Three different flow rates were used: 0.8ml/min, 1.0ml/min and 1.2ml/min, and the separation effect and the content of the terpene lactones are examined. The results show that of the three different flow rates, the flow rate of 1.0ml/min has higher terpene lactone content and proper separation degree and retention time, so the flow rate of 1.0ml/min is selected.

Investigation of column temperature

Three different column temperatures were used: the separation effect and content of terpene lactones were examined at 25 deg.C, 30 deg.C, and 35 deg.C. The results show that the separation degree and retention time of terpene lactones are more suitable when the temperature is 30 ℃ in three different column temperatures, so that the column temperature is selected to be 30 ℃.

Methodology survey

3.3.1. Investigation of linear relationships

Taking a ginkgolide C reference substance, a bilobalide A reference substance, a ginkgolide B reference substance respectively to prepare and dilute into 6 gradient concentrations, measuring peak areas, taking the natural logarithm (lnA) of the peak area A as a vertical coordinate, taking the natural logarithm (lnC) of the sample amount C (mug) as a horizontal coordinate, and performing linear regression calculation, wherein the linear regression equation of the ginkgolide C is as follows: lnA =1.7099lnC + 5.1638, r = 0.9993, the linear regression equation of bilobalide is: lnA =1.6903lnC + 5.2922, r = 0.9993; the linear regression equation of ginkgolide A is as follows: lnA =1.6735lnC + 4.7526, r = 0.9994, and the linear regression equation for ginkgolide B is: lnA =1.71lnC + 4.3577, r = 0.9994.

3.3.2. Precision survey

Sampling, preparing a sample according to a preparation method of a text sample solution, continuously sampling for 6 times, and recording a chromatogram, wherein the result shows that the peak area RSD of the ginkgolide C is 1.37%; the peak area RSD of the bilobalide is 1.40 percent; the RSD of the ginkgolide A peak area is 1.25 percent, the RSD of the ginkgolide B peak area is 1.06 percent, and the precision of the instrument is good.

3.3.3. Repeatability test

Sampling, preparing 6 parts of test sample according to the preparation method of the test sample solution in the text, injecting the test sample into a liquid chromatograph, and recording a chromatogram, wherein the average value of the total amount of terpene lactones is 0.272mg/ml, and the RSD is 1.81 percent, which indicates that the method has good repeatability.

3.3.4. Stability test

Taking a sample, preparing a test solution according to the text test solution preparation method, and respectively measuring the peak areas of ginkgolide C, bilobalide, ginkgolide A and ginkgolide B at 0,2, 4, 6, 12 and 24 hours, wherein the result shows that the test solution has good stability within 24 hours.

3.3.5. Recovery test

By adopting a sample-adding recovery method, the average recovery rate of the ginkgolide C is 94.82 percent, and the RSD is 2.38 percent; the average recovery rate of the bilobalide is 106.66 percent, and the RSD is 2.3 percent; the average recovery rate of the ginkgolide A is 97.89%, the RSD is 4.38%, the average recovery rate of the ginkgolide B is 97.73%, and the RSD is 4.83%.

3.3.6. Sample assay

The content of 30 batches of ginkgo dipyridamole injection is measured, and the result is obtainedThe total amount of terpene lactone is 0.250-0.322 mg/ml, and the average amount is 0.272mg/ml, so that every 1ml contains terpene lactone and bilobalide (C) ₁₅ H ₁₈ O ₈ ) Ginkgolide A (C) ₂₀ H ₂₄ O ₉ ) Ginkgolide B (C) ₂₀ H ₂₄ O ₁₀ ) And ginkgolide C (C) ₂₀ H ₂₄ O ₁₁ ) The total amount is not less than 0.18 mg.

The specific embodiment is as follows:

example 1:

the detection method of the ginkgo dipyridamole injection comprises the following steps:

the characteristics are as follows:

the product is a clear liquid with yellow to brown yellow;

and (3) identification:

(1) taking 2ml of the product, adding a little magnesium powder, adding a plurality of drops of concentrated hydrochloric acid, and standing to be gradually red;

(2) taking 2ml of the product, adding 10ml of ethanol to show green fluorescence, and adding diluted hydrochloric acid to remove the fluorescence;

(4) taking 25ml of the product, passing through D201 type macroporous adsorbent resin column (inner diameter of 2cm, column height of 25 cm), eluting with 200ml of water, discarding water washing solution, eluting with 200ml of ethanol, collecting eluate, evaporating to dryness, adding 20ml of n-butanol into residue, soaking in water bath for 20 min while shaking, cooling, filtering, evaporating filtrate to dryness, and adding 1ml of ethanol into residue to dissolve to obtain sample solution. Taking folium Ginkgo control extract 0.1g, adding n-butanol 20ml, placing in water bath, soaking for 20 minutes while shaking, cooling, filtering, evaporating filtrate to dryness, and dissolving residue with ethanol 1ml to obtain control extract solution. According to the test of Chinese pharmacopoeia thin-layer chromatography, 2 mul of each of the test solution and the control extract solution are respectively spotted on the same silica gel G thin-layer plate which takes sodium carboxymethyl cellulose solution containing 4% of sodium acetate as a binder, and the weight ratio of ethyl acetate: butanone: formic acid: water 5: 4: 0.5: developing with developer 1, taking out, air drying, and spraying with 5% aluminum trichloride ethanol solution. Inspecting under 365nm ultraviolet lamp. Fluorescent spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control extract;

and (4) checking:

(3) precisely measuring the residues on the red skin to obtain 10ml of ginkgo dipyridamole injection, evaporating to dry, and checking according to the residue on red skin inspection method of Chinese pharmacopoeia that each 1ml of the injection does not exceed 1.0 mg;

(4) accurately weighing 10ml of the total solid ginkgo dipyridamole injection, placing the ginkgo dipyridamole injection in an evaporation dish dried to constant weight, after evaporating to dryness in a water bath, accurately adding 3g of diatomite dried to constant weight at 105 ℃, stirring, drying for 3 hours at 105 ℃, moving the ginkgo dipyridamole injection in a dryer, cooling for 30 minutes, quickly and accurately weighing the weight, deducting the amount of the added diatomite, and calculating that the total solid content in every 1ml of the injection is 0.2-0.3 g;

chromatographic condition and system adaptability test takes octadecylsilane chemically bonded silica as a filler, methanol: 0.4% glacial acetic acid ═ 4:96 is a mobile phase, the flow rate is 1.0ml for each species, the column temperature is 30 ℃, and the detection wavelength is 284 nm; the number of theoretical plates is not less than 4000 calculated according to the peak of 5-hydroxymethylfurfural; preparing a reference substance solution, precisely weighing a proper amount of 5-hydroxymethylfurfural reference substance, and adding methanol to prepare a solution containing 50 mu g of methanol per lml to serve as the reference substance solution;

the determination method comprises precisely sucking 10 μ l of each of the reference solution and the test solution, injecting into a liquid chromatograph, and recording chromatogram; calculating the content of 5-hydroxymethylfurfural;

0-30min, mobile phase A75 → 90%, mobile phase B25 → 10%;

30-35min, mobile phase A90%, mobile phase B10%;

25-35min, mobile phase A17%, mobile phase B83%;

35-36min, mobile phase A90 → 75%, mobile phase B10 → 25%;

36-45min, mobile phase A75%, mobile phase B25%;

preparation of reference solution A suitable amount of neoacid ginkgolide reference was weighed precisely and added with methanol to make 1 μ g solution per 1ml as reference solution. And preparing a proper amount of total ginkgoic acid reference substance into a solution containing 20 mu g of total ginkgoic acid per 1ml by using methanol, wherein the solution is used as a reference solution for positioning.

the determination method precisely absorbs 50 μ l of each of the test solution, the reference solution and the positioning reference solution, injects into the liquid chromatograph, and records the chromatogram; calculating the total peak area of the corresponding chromatographic peak of the total ginkgoic acid reference substance in the test solution, and calculating the content of the total ginkgoic acid by using a ginkgolic neo-acid reference substance external standard method;

(9) the tannin is determined by checking with Chinese pharmacopoeia injection, taking 1ml of the product, adding newly prepared physiological sodium chloride solution containing 1% egg white 5ml [ if necessary, filtering with microporous membrane (0.45 μm) ], standing for 10 min, and no turbidity or precipitate appears. If turbidity or precipitation occurs, taking the injection lml, adding 1 drop of dilute acetic acid, and then adding 4-5 drops of sodium chloride gelatin test solution to avoid turbidity or precipitation;

(10) the oxalate is determined according to a Chinese pharmacopoeia injection related substance inspection method, 5ml of the oxalate is taken, the pH value is adjusted to 1-2 by using dilute hydrochloric acid, filtration is carried out, 2ml of filtrate is taken, the pH value of the filtrate is adjusted to 5-6, 2-3 drops of 3% calcium chloride solution are added, and the mixture is placed for 10 minutes without turbidity or precipitation;

(11) the resin is determined according to the inspection method of related substances of Chinese pharmacopoeia injection, 5ml of the product is taken, 1 drop of hydrochloric acid is added, and the product is placed for 30 minutes without precipitation. If precipitation occurs, adding 10ml of chloroform into 5ml of the product, shaking for extraction, collecting chloroform solution, evaporating in water bath, dissolving the residue with 2ml of glacial acetic acid, placing in a test tube with a plug, adding 3ml of water, mixing, and standing for 30min to obtain no precipitation;

(12) measuring potassium ions according to a related substance inspection method of Chinese pharmacopoeia injection, taking 2ml of the product, evaporating to dryness, burning with small fire until charring, burning at 500-600 ℃ until completely incinerating, adding 2ml of dilute acetic acid for dissolving, placing in a 25ml measuring flask, adding water for diluting to a scale, and mixing uniformly to obtain a test solution. Taking two 10ml Nashi colorimetric tubes, precisely adding 0.8ml of standard potassium ion solution into the tube A, adding 0.6ml of alkaline formaldehyde solution (taking the formaldehyde solution, adjusting the pH value to 8.0-9.0 by using 0.1mol/L sodium hydroxide solution) and 2 drops of 3% ethylenediamine tetraacetate disodium solution and 0.5ml of 3% sodium tetraphenylborate solution, adding water to dilute the formaldehyde solution into 10ml, precisely adding a sample solution lml into the tube B, performing operation according to the method simultaneously with the tube A, shaking uniformly, placing the tube A and the tube B on black paper, performing perspective from top to bottom, and preventing the turbidity displayed in the tube B from being more concentrated compared with the tube A;

(13) diluting the product with 10% glucose injection for one time in pyrogen, and checking according to Chinese pharmacopoeia pyrogen checking method, wherein the dosage is 3ml according to rabbit weight 1 kg slowly injecting diluent;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; performing gradient elution by using acetonitrile as a mobile phase A and 0.4% phosphoric acid solution as a mobile phase B; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 254 nm; the number of theoretical plates is not less than 5000 according to rutin peak;

the gradient elution conditions were:

0-5min, mobile phase A8 → 12%, mobile phase B92 → 88%;

5-25min, mobile phase A12 → 17%, mobile phase B88 → 83%;

25-35min, mobile phase A17%, mobile phase B83%;

35-45min, mobile phase A17 → 23%, mobile phase B83 → 77%;

45-60min, mobile phase A23 → 25%, mobile phase B77 → 75%;

60-75min, mobile phase A25 → 60%, mobile phase B75 → 40%;

preparing reference solution by accurately weighing rutin reference substance, and adding methanol to obtain 0.1 mg/1 ml solution to obtain reference solution;

the determination method precisely absorbs 10 μ l of each of the reference solution and the sample solution, injects into a liquid chromatograph, determines, and records chromatogram for 75 minutes to obtain the sample fingerprint;

the test sample fingerprint should have 15 common peaks, should present chromatographic peak with same retention time as reference substance chromatographic peak, common peak match, calculate according to Chinese medicine chromatogram fingerprint similarity evaluation system, the similarity of the test sample fingerprint and the reference fingerprint must not be lower than 0.90;

content determination:

chromatographic conditions and system suitability test using octadecylsilane chemically bonded silica as a filler, 0.1% sodium dihydrogen phosphate solution [ pH adjusted to 4.6 with (1 → 3) phosphoric acid solution ]: methanol 26: 74 is a mobile phase; the flow rate was 1.0ml per minute; the detection wavelength is 290 nm; the number of theoretical plates is not less than 2000 calculated according to dipyridamole peak;

preparation of reference solution A proper amount of dipyridamole reference was precisely weighed, and 80% methanol solution was added to make a solution containing 15 μ g of dipyridamole per 1ml as a reference solution;

precisely weighing 2ml of the product for sample solution preparation, placing into a 50ml measuring flask, adding 80% methanol solution to dilute to scale, and shaking to obtain sample solution;

precisely measuring 10 μ l of reference solution and sample solution by determination method, respectively injecting into liquid chromatograph, recording chromatogram, and calculating dipyridamole content by peak area according to external standard method;

the content of dipyridamole in 1ml of the product should be 0.36-0.44 mg.

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; the method comprises the following steps of mixing methanol: 0.2% phosphoric acid solution ═ 48: 52 is a mobile phase; the flow rate was 1.0ml per minute; the column temperature is 20 ℃; the detection wavelength is 360 nm; the theoretical plate number is not lower than 2500 calculated according to the peak of quercetin;

preparation of reference substance solution A proper amount of quercetin reference substance, kaempferide reference substance, and isorhamnetin reference substance are precisely weighed, methanol is added to prepare a mixed solution containing 0.03mg of quercetin, 0.025mg of kaempferide, and 0.01mg of isorhamnetin per 1ml, and the mixed solution is shaken up to obtain a reference substance solution;

preparation of test solution 5ml of the product was precisely transferred, and methanol was added: 33% hydrochloric acid solution ═ 6: 1, heating and refluxing the mixed solution on a water bath for 90 minutes, quickly cooling to room temperature, transferring to a 50ml measuring flask, diluting to a scale with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a test solution;

the total flavonol glycoside content of the product should be 0.85-1.10 mg per 1 ml.

(3) The rutin, kaempferol-3-O-rutinoside and narcissus glycoside are measured according to the high performance liquid chromatography of Chinese pharmacopoeia:

in chromatographic condition and system applicability test, octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.4% phosphoric acid solution (15: 85) is used as a mobile phase; flow rate 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 360 nm; the theoretical plate number is not less than 5000 calculated according to rutin peak;

measuring by accurately sucking 10 μ l of reference solution and test solution respectively, injecting into liquid chromatograph, recording chromatogram, and calculating rutin, kaempferol-3-O-rutinoside and narcissus glycoside content respectively;

the product contains rutin (C) per 1ml ₂₇ H ₃₀ O ₁₆ ) 0.06-0.12 mg of kaempferol-3-O-rutinoside (C) ₂₇ H ₃₀ O ₁₅ ) 0.05mg to 0.10mg of hydrastin (C) ₂₈ H ₃₂ O ₁₆ ) The amount of the active ingredient is 0.04-0.08 mg.

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; mixing the raw materials in a ratio of n-propanol: tetrahydrofuran (tetrahydrofuran): water is a mobile phase with the ratio of 1:14: 85; detecting with an evaporative light scattering detector; the number of theoretical plates is not less than 2500 calculated according to the peak of bilobalide;

preparing a test solution by precisely measuring 25ml of the product, adding 2 drops of 2% hydrochloric acid solution, shaking and extracting with ethyl acetate for 5 times, 30ml for the 1 st time and 20ml for the other 4 times, combining ethyl acetate extract, washing with 20ml of 5% sodium acetate solution, separating sodium acetate solution, and washing with 200ml of ethyl acetate; mixing the ethyl acetate extractive solution and washing solution, washing with water for 2 times, each time 20ml, separating water solution, washing with ethyl acetate 10ml, mixing ethyl acetate solutions, recovering solvent to dryness, dissolving residue with methanol, transferring to 2ml measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate to obtain sample solution;

and precisely sucking 3 mu l and 10 mu l of the reference extract solution and 5-10 mu l of the test solution respectively by a determination method, injecting the reference extract solution and the test solution into a liquid chromatograph, determining, and respectively calculating the contents of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C by using an external standard two-point method logarithmic equation.

Every 1ml injection contains bilobalide (C) ₁₅ H ₁₈ O ₈ ) Ginkgolide A (C) ₂₀ H ₂₄ O ₉ ) Ginkgolide B (C) ₂₀ H ₂₄ O ₁₀ ) And ginkgolide C (C) ₂₀ H ₂₄ O ₁₁ ) The total amount should not be less than 0.18 mg.

Example 2:

the characteristics are as follows:

the product is a clear liquid with yellow to brown yellow;

and (3) identification:

(1) taking 2ml of the product, adding a little magnesium powder, adding a plurality of drops of concentrated hydrochloric acid, and gradually developing red after standing;

(4) taking 50ml of the product, passing through a D101 type macroporous adsorption resin column (with an inner diameter of 2cm and a column height of 27cm), eluting with 250ml of water, discarding a water washing solution, eluting with 250ml of ethanol, collecting an eluent, evaporating to dryness, adding 15ml of n-butanol into residues, soaking in a water bath for 15 minutes at a warm temperature, shaking at any time, cooling, filtering, evaporating to dryness, adding 2ml of ethanol into residues, dissolving, and taking the residues as a test solution. Taking folium Ginkgo control extract 0.2g, adding n-butanol 15ml, placing in water bath, soaking for 15 minutes while shaking constantly, cooling, filtering, evaporating filtrate to dryness, and dissolving residue with ethanol 2ml to obtain control extract solution. According to the test of Chinese pharmacopoeia thin-layer chromatography, 1 microliter of each of the test solution and the control extract solution is respectively spotted on the same silica gel G thin-layer plate which takes sodium carboxymethyl cellulose solution containing 4% of sodium acetate as a binder, and the weight ratio of ethyl acetate: butanone: formic acid: water 5: 3: 1: developing with developing agent 1, taking out, air drying, and spraying with 3% aluminum trichloride ethanol solution. Inspecting under 365nm ultraviolet lamp. Fluorescent spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control extract;

and (4) checking:

(5) the 5-hydroxymethylfurfural is determined by high performance liquid chromatography in Chinese pharmacopoeia, octadecylsilane chemically bonded silica is used as a filler (Agilent ZORBAX extended C18, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m), and methanol: 0.4% glacial acetic acid ═ 4:96 is a mobile phase, the flow rate is 1.0ml for each species, the column temperature is 30 ℃, and the detection wavelength is 284 nm; the number of theoretical plates is not less than 4000 calculated according to the peak of 5-hydroxymethylfurfural; taking a proper amount of 5-hydroxymethylfurfural reference substance, precisely weighing, and adding methanol to prepare a solution containing 50 mu g of 5-hydroxymethylfurfural per lml as a reference substance solution; filtering semen Ginkgo dipyridamole injection to obtain filtrate as sample solution; accurately sucking 10 μ l of each of the reference solution and the test solution, injecting into a liquid chromatograph, and recording chromatogram; calculating the content of 5-hydroxymethylfurfural; the product contains 5-hydroxymethylfurfural in an amount of 0.01 mg/1 ml.

(6) The residual quantity of heavy metal and harmful elements is measured by a lead, cadmium, arsenic, mercury and copper measuring method (2321 atomic absorption spectrophotometry in the pharmacopoeia 2015 of China, the version rule) and lead is not more than 0.24 mu g/ml; cadmium is not more than 0.06 mu g/ml; arsenic is not more than 0.12 mu g/ml; the mercury content is not more than 0.04 mu g/ml; the copper content should not exceed 3.00. mu.g/ml.

(7) Diluting the product with 10% glucose injection for one time, and checking according to Chinese pharmacopoeia pyrogen check method, wherein the dosage is 3ml of rabbit weight 1 kg slowly injecting diluent, and should meet the regulation;

(8) other should accord with the relevant regulations under the Chinese pharmacopoeia injection item;

content determination:

chromatographic conditions and system applicability test using octadecylsilane chemically bonded silica as a filler, 0.1% sodium dihydrogen phosphate solution [ pH adjusted to 4.6 with (1 → 3) phosphoric acid solution ]: methanol 26: 74 is a mobile phase; the flow rate was 1.0ml per minute; the detection wavelength is 290 nm; the number of theoretical plates is not less than 2000 calculated according to dipyridamole peak;

preparing a test solution, precisely measuring 2ml of the product, placing the product in a 50ml measuring flask, adding 80% methanol solution to dilute the product to a scale, and shaking up to obtain the test solution;

precisely measuring 20 μ l of reference solution and sample solution by determination method, respectively injecting into liquid chromatograph, recording chromatogram, and calculating dipyridamole content by peak area according to external standard method;

the content of dipyridamole in 1ml of the product should be 0.36-0.44 mg.

in chromatographic condition and system applicability test, octadecylsilane chemically bonded silica is used as a filler; the method comprises the following steps of mixing methanol: 0.4% phosphoric acid solution ═ 50: 50 is a mobile phase; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 360 nm; the theoretical plate number is not lower than 2500 calculated according to the peak of quercetin;

preparation of test solution 10ml of the product was precisely transferred, and methanol was added: 33% hydrochloric acid solution ═ 6: 1, heating and refluxing the mixed solution on a water bath for 60 minutes (the flow temperature is 85 ℃), quickly cooling to room temperature, transferring to a 50ml measuring flask, diluting to a scale with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a test solution;

the determination method precisely absorbs 10 μ l of each of the reference solution and the test solution, injects into a liquid chromatograph, records chromatogram, and calculates the total flavonol glycoside content according to the following formula:

the total flavonol glycosides contained in 1ml of the product should be 0.85-1.10 mg.

(3) The terpene lactones are measured by high performance liquid chromatography according to Chinese pharmacopoeia:

preparing a test solution by precisely measuring 20ml of the product, adding 2 drops of 2% hydrochloric acid solution, shaking and extracting with ethyl acetate for 5 times, 20ml for the 1 st time, and 15ml for the other 4 times, combining ethyl acetate extract, washing with 20ml of 5% sodium acetate solution, separating sodium acetate solution, and washing with 10ml of ethyl acetate; mixing the ethyl acetate extractive solution and the washing solution, washing with water for 2 times (20 ml each time), separating water solution, washing with ethyl acetate 10ml, mixing ethyl acetate solutions, recovering solvent to dry, dissolving the residue with methanol, transferring to 2ml measuring flask, adding methanol to scale, shaking, filtering, and collecting filtrate to obtain sample solution;

The product contains bilobalide (C) and ginkgolide in 1ml injection ₁₅ H ₁₈ O ₈ ) Ginkgolide A (C) ₂₀ H ₂₄ O ₉ ) Ginkgolide B (C) ₂₀ H ₂₄ O ₁₀ ) And ginkgolide C (C) ₂₀ H ₂₄ O ₁₁ ) The total amount should not be less than 0.18 mg.

Description of the drawings:

FIG. 1 shows the fingerprint of Ginkgo dipyridamole injection

Peak 4: sphenin peak 6: rutin peak 9: quercetin 3-O-glucosyl (1 → 2) rhamnoside

Peak 10: kaempferol-3-O-rutinoside peak 12: narcissus glycosides

Peak 13: 3-O- {2-O- [6-O- (p-hydroxy-trans-coumaroyl) -glucosyl ] -rhamnosyl } quercetin

Peak 14: 3-O- {2-O- [6-O- (p-hydroxy-trans-coumaroyl) -glucosyl ] -rhamnosyl } kaempferol

Peak 15: dipyridamole.

Claims

1. A detection method of a ginkgo dipyridamole injection preparation, which is an injection prepared from ginkgo biloba extract and dipyridamole, is characterized in that: the detection method comprises the following steps:

the characteristics are as follows:

the injection is yellow to brown yellow clear liquid;

and (3) identification:

(4) taking 50ml of injection, passing through D101 type macroporous adsorbent resin column, eluting with 250ml of water, discarding water eluate, eluting with 250ml of ethanol, collecting eluate, evaporating to dryness, adding 15ml of n-butanol into residue, soaking in water bath for 15 min, shaking, cooling, filtering, evaporating filtrate to dryness, and dissolving residue with 2ml of ethanol to obtain sample solution; collecting folium Ginkgo control extract 0.2g, adding n-butanol 15ml, and preparing control extract solution by the same method; according to the test of Chinese pharmacopoeia thin-layer chromatography, 1 mu l of each of the two solutions is sucked and respectively spotted on the same silica gel G thin-layer plate which takes sodium carboxymethyl cellulose solution containing 4% sodium acetate as a binder, and the weight ratio of ethyl acetate: butanone: formic acid: water 5: 3: 1:1 is developing agent, taking out, airing, and spraying 3% aluminum trichloride ethanol solution; inspecting under 365nm ultraviolet lamp; fluorescent spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control extract;

and (3) checking:

the pH value is checked according to a pH value measuring method of Chinese pharmacopoeia, and the pH value is 3.5-5.5;

the peak area ratio of the flavone aglycone is calculated according to the chromatogram under the content measurement item of the total flavonol glycoside, the peak area ratio of the quercetin to the kaempferide is 0.8-1.2, and the peak area ratio of the isorhamnetin to the quercetin is more than 0.15;

precisely measuring 10ml of injection for residues on ignition, evaporating to dryness, and checking according to residue on ignition check method in Chinese pharmacopoeia, wherein each 1ml of the residue on ignition should not exceed 1.0 mg;

precisely measuring 10ml of total solid, placing the injection in an evaporating dish dried to constant weight, evaporating in a water bath, precisely adding 3g of diatomite dried to constant weight at 105 ℃, stirring, placing the mixture in a drier for drying for 3 hours at 105 ℃, cooling for 30 minutes, rapidly and precisely weighing the weight, deducting the amount of the added diatomite, and calculating that the total solid content in each 1ml of the injection is 0.2-0.3 g;

the 5-hydroxymethylfurfural is measured according to the high performance liquid chromatography of Chinese pharmacopoeia:

the total ginkgoic acid is measured according to the high performance liquid chromatography of Chinese pharmacopoeia:

performing gradient elution under chromatographic conditions and system adaptability test by using octadecylsilane chemically bonded silica as filler, acetonitrile containing 0.1% trifluoroacetic acid as mobile phase A and water containing 0.1% trifluoroacetic acid as mobile phase B, wherein the detection wavelength is 310nm, and the number of theoretical plates is not less than 4000 according to the peak of ginkgolic acid; the gradient elution conditions were:

0-30min, mobile phase A75 → 90%, mobile phase B25 → 10%;

30-35min, mobile phase A90%, mobile phase B10%;

25-35min, mobile phase A17%, mobile phase B83%;

35-36min, mobile phase A90 → 75%, mobile phase B10 → 25%;

36-45min, mobile phase A75%, mobile phase B25%;

preparing reference solution by accurately weighing appropriate amount of neoacid of semen Ginkgo, adding methanol to obtain 1 μ g solution per 1ml as reference solution, taking appropriate amount of total ginkgolic acid reference solution, preparing 20 μ g solution per 1ml with methanol as reference solution for positioning,

the determination method comprises precisely sucking sample solution, reference solution and positioning reference solution 50 μ l respectively, injecting into liquid chromatograph, and recording chromatogram; calculating the total peak area of the corresponding chromatographic peak of the total ginkgoic acid reference substance in the test solution, and calculating the content of the total ginkgoic acid by using a ginkgolic neo-acid reference substance external standard method;

the residual amounts of heavy metals and harmful elements are determined by the determination method of lead, cadmium, arsenic, mercury and copper in Chinese pharmacopoeia, and each 1ml of injection contains lead of 0.24 mu g, cadmium of 0.06 mu g, arsenic of 0.12 mu g, mercury of 0.04 mu g and copper of 3.00 mu g;

the protein is measured according to a Chinese pharmacopoeia injection related substance inspection method, 1ml of the product is taken, a newly prepared 30% sulfosalicylic acid solution lml is added, the mixture is uniformly mixed and placed for 5 minutes, no turbidity is generated, and if a component generating precipitation by acid exists in the injection, 1-3 drops of tannic acid test solution is added, no turbidity is generated;

the tannin is determined according to a Chinese pharmacopoeia injection related substance inspection method, 1ml of the product is taken, 5ml of newly prepared physiological sodium chloride solution containing 1% egg white is added, the mixture is placed for 10 minutes, no turbidity or precipitation is caused, if turbidity or precipitation is caused, 1 drop of dilute acetic acid is added into the injection lml, 4-5 drops of sodium chloride gelatin test solution are added, and no turbidity or precipitation is caused;

the oxalate is determined according to a Chinese pharmacopoeia injection related substance inspection method, 5ml of the oxalate is taken, the pH value is adjusted to 1-2 by using dilute hydrochloric acid, filtration is carried out, 2ml of filtrate is taken, the pH value of the filtrate is adjusted to 5-6, 2-3 drops of 3% calcium chloride solution are added, and the mixture is placed for 10 minutes without turbidity or precipitation;

the resin is determined according to the inspection method of related substances of Chinese pharmacopoeia injection, 5ml of the product is taken, 1 drop of hydrochloric acid is added, the product is placed for 30 minutes, no precipitation is generated, if precipitation is generated, 5ml of the product is taken, 10ml of trichloromethane is added, shaking extraction is carried out, trichloromethane liquid is taken separately, the trichloromethane liquid is placed on a water bath and dried by distillation, 2ml of glacial acetic acid is added to residues for dissolution, the trichloromethane liquid is placed in a test tube with a plug, 3ml of water is added, the mixture is mixed evenly, and the resin is placed for 30 minutes, no precipitation is generated;

measuring potassium ions according to a Chinese pharmacopoeia injection related substance inspection method, taking 2ml of the product, evaporating to dryness, burning with small fire until charring, burning with 500-600 ℃ until completely incinerating, adding 2ml of dilute acetic acid for dissolving, placing in a 25ml measuring flask, adding water for diluting to a scale, uniformly mixing to obtain a test solution, taking two 10ml Nashi colorimetric tubes, precisely adding 0.8ml of a standard potassium ion solution into the A tube, adding 0.6ml of an alkaline formaldehyde solution, 2 drops of a 3% ethylenediamine tetraacetate disodium solution and 0.5ml of a 3% sodium tetraphenylborate solution into the A tube, adding water for diluting to 10ml, precisely adding a test solution lml into the B tube, operating with the A tube simultaneously according to the method, shaking uniformly, placing the A tube and the B tube on black paper together, performing perspective from top to bottom, and comparing turbidity generated in the B tube with the A tube to obtain no more concentration; the alkaline formaldehyde solution is prepared by taking a formaldehyde solution, and adjusting the pH value to 8.0-9.0 by using 0.1mol/L sodium hydroxide solution;

diluting the pyrogen injection with 10% glucose injection by one time, examining according to Chinese pharmacopoeia pyrogen examination method, and slowly injecting 3ml of diluent with a dosage of 1 kg rabbit weight, wherein the dosage is in accordance with the regulation;

other should accord with Chinese pharmacopoeia injection item relevant regulation;

the gradient elution conditions were:

0-5min, mobile phase A8 → 12%, mobile phase B92 → 88%;

5-25min, mobile phase A12 → 17%, mobile phase B88 → 83%;

25-35min, mobile phase A17%, mobile phase B83%;

35-45min, mobile phase A17 → 23%, mobile phase B83 → 77%;

45-60min, mobile phase A23 → 25%, mobile phase B77 → 75%;

60-75min, mobile phase A25 → 60%, mobile phase B75 → 40%;

the sample fingerprint should have chromatographic peaks with the same retention time as the reference chromatographic peaks, and there should be 15 common peaks, wherein the peak No. 4 is a pterosin peak, the peak No. 6 is a rutin peak, the peak No. 9 is a quercetin 3-O-glucosyl (1 → 2) rhamnoside peak, the peak No. 10 is a kaempferol-3-O-rutinoside peak, the peak No. 11 is a narcissus peak, the peak No. 13 is a 3-O- {2-O- [6-O- (p-hydroxy-trans-coumaroyl) -glucosyl ] -rhamnosyl } quercetin peak, the peak No. 14 is a 3-O- {2-O- [6-O- (p-hydroxy-trans-coumaroyl) -glucosyl ] -rhamnosyl } kaempferol peak, and the peak No. 15 is a dipyridamole peak; common peak matching, which is calculated according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and the similarity between the fingerprint of the test sample and the fingerprint of the reference sample is not less than 0.90;

content determination:

chromatographic conditions and system suitability test octadecylsilane chemically bonded silica was used as a filler, and a solution of 0.1% sodium dihydrogen phosphate: 25 parts of methanol: 75 is a mobile phase, and the 0.1 percent sodium dihydrogen phosphate solution is adjusted to the pH value of 4.6 by a 1 → 3 phosphoric acid solution in advance; the flow rate was 1.0ml per minute; the detection wavelength is 290 nm; the number of theoretical plates is not less than 2000 calculated according to dipyridamole peak;

preparing a test solution, precisely measuring 2ml of injection, placing the injection into a 50ml measuring flask, adding 80% methanol solution to dilute to a scale, shaking up, and taking a subsequent filtrate to obtain the test solution;

in chromatographic condition and system applicability test, octadecylsilane chemically bonded silica is used as a filler; with acetonitrile-0.4% phosphoric acid solution = 16: 84 is a mobile phase; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 360 nm; the theoretical plate number is not less than 5000 according to rutin peak;

preparation of reference substance solution A proper amount of rutin reference substance, kaempferol-3-O-rutinoside reference substance and narcissus reference substance are precisely weighed, 80% methanol is added to prepare a mixed solution containing 0.08mg of rutin, 0.07mg of kaempferol-3-O-rutinoside and 0.06mg of narcissus in each 1ml, and the mixed solution is shaken up to obtain a reference substance solution;

in chromatographic condition and system applicability test, octadecylsilane chemically bonded silica is used as a filler; mixing the raw materials in a ratio of n-propanol: tetrahydrofuran (tetrahydrofuran): water 1:15:84 is mobile phase; detecting with an evaporative light scattering detector; the number of theoretical plates is not less than 2500 calculated according to the peak of bilobalide;

2. The detection method according to claim 1, characterized in that: among the examination items: the 5-hydroxymethylfurfural limit is as follows: the content of 5-hydroxymethylfurfural in each 1ml of injection is not more than 0.01 mg; the total ginkgoic acid limit is as follows: each 1ml of injection contains no more than 15ng of total ginkgoic acid; in the content measurement items: every 1ml of injection contains 0.36-0.44 mg of dipyridamole; the total flavonol glycoside content of each 1ml injection is 0.85-1.10 mg; each 1ml of injection contains 0.06mg to 0.12mg of rutin, 0.05mg to 0.10mg of kaempferol-3-O-rutinoside and 0.04mg to 0.08mg of hydrangeaside; terpene lactone contained in 1ml injection is not less than 0.18mg (based on total amount of bilobalide, bilobalide A, bilobalide B and bilobalide C).