CN1939391A

CN1939391A - Quality control of Chinese-medicine compound preparation

Info

Publication number: CN1939391A
Application number: CNA2005101078199A
Authority: CN
Inventors: 于文风
Original assignee: Qiyuanyide Medicines Institute Beijing
Current assignee: Qiyuanyide Medicines Institute Beijing
Priority date: 2005-09-30
Filing date: 2005-09-30
Publication date: 2007-04-04

Abstract

A quality control method for the Chinese medicine prepared from astragalus root and notoginseng or its extract includes fingerprint test method and/or differentiation test method and/or content measuring method.

Description

A kind of method of quality control of compound Chinese medicinal preparation

Technical field

The present invention is a kind of method of quality control of compound Chinese medicinal preparation, belongs to technical field of Chinese medicine.

Background technology

Cardiovascular and cerebrovascular disease such as coronary heart disease, cerebral thrombosis, alzheimer disease etc. all are one of diseases that the world today is the most common and harm is maximum, have become human mortality's one of the main reasons in many countries; According to investigations, sickness rate in recent years has and increases trend year by year, and in, young patient constantly increases, ischemic cardiovascular and cerebral vascular disease has become commonly encountered diseases, the frequently-occurring disease of harm China people ' s health; Prevent and treat purpose in order to reach, number of research projects has been done to it by many inventors and medicine enterprise.Pharmaceutical preparation must be on the basis that guarantees the constant product quality controllable safety, constantly more new development, in order better to control the quality of said preparation, guarantee the safety of medication, better instruct and produce, make technology controlling and process rationally strict more, make consumer's energy full appreciation product quality, need research, control this compound Chinese medicinal preparation method for quality; At present, in the related drugs preparation, generally only with astragaloside, Panax Notoginseng saponin R ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁Be to detect index, but its quality of reactor product comprehensively at all not very reasonable with this quality that is used for controlling compound Chinese medicinal preparation only; If control,, relatively be difficult to implement owing to do not have ready-made detection scheme, testing conditions or the like with other index.

Summary of the invention

The object of the present invention is to provide a kind of method of quality control of new compound Chinese medicinal preparation.This method provides means, technical method of the index that detects, detection or the like to relevant production, testing agency, so that better control the quality of said preparation, guarantee the safety of medication, can better instruct production, make controlling of production process rationally strict more, make consumer's energy full appreciation product quality.

The flavour of a drug of this compound Chinese medicinal preparation are formed and proportioning following (by weight):

Scheme one:

Radix Notoginseng 1～99 weight portion Radix Astragali 99～1 weight portions

Scheme two:

Radix Notoginseng 1～99 weight portion Radix Astragali total saponins 30～0.1 weight portions

Scheme three:

Radix Notoginseng total arasaponins 0.1～30 weight portion Radix Astragali 99～1 weight portions

Scheme four:

Radix Notoginseng total arasaponins 0.1～30 weight portion Radix Astragali total saponins 30～0.1 weight portions

The dosage form of above-mentioned compound Chinese medicinal preparation is injection or oral formulations; Wherein injection comprises: be directly used in the injection of drug administration by injection, directly for the venous transfusion of intravenous drip, need to be used for after the dilution concentrated solution for injection of intravenous drip and with the injectable sterile powder or the aseptic block of freeze-drying or spray drying method for preparation; Oral formulations comprises tablet, dispersible tablet, capsule, soft capsule, microcapsule, granule, pill, pellet, powder, drop pill, slow releasing preparation, controlled release preparation, oral liquid, gel, soft extract, extractum and membrane.Be used for the treatment of diseases such as coronary heart disease, angina pectoris, arrhythmia, cerebral thrombosis, alzheimer disease, hepatorenal syndrome, heart and lung diseases, diabetes and complication thereof clinically.

The present invention constitutes like this:

The method of quality control of the compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng, it is characterized in that: this method comprises following all or part of content:

(1) finger printing test comprises with the Radix Astragali flavone constituents being characterized as main finger printing and being characterized as in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents;

(2) Milkvetch Root, Radix Notoginseng control medicinal material, astragaloside, formononetin, calycosin, Panax Notoginseng saponin R ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁In the differential test method of all or part of composition;

(3) astragaloside, formononetin, calycosin, total saponins, Panax Notoginseng saponin R ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁In the content test method of all or part of composition.

The method of quality control of the compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng is characterized in that: this method adopts liquid chromatography test to be characterized as main finger printing and to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents:

A, the test of employing liquid chromatography are characterized as main finger printing with the Radix Astragali flavone constituents:

(1) preparation of need testing solution: it is an amount of to get compound Chinese medicinal preparation to be measured, adds the dissolving of water or methanol or ethanol or n-butyl alcohol, filters, and filtrate evaporate to dryness, residue add methanol or dissolve with ethanol or be diluted to suitable concn, as need testing solution;

(2) preparation of object of reference solution: get formononetin,, be settled to suitable concn, as object of reference solution with methanol or dissolve with ethanol;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol: water or 0.1%～5% glacial acetic acid or 0.1%～5% formic acid or 0.005%～5% phosphoric acid solution, gradient elution, flow velocity is that 0.5～2.0ml/min, detection wavelength are one or several in the 190-400nm scope, and column temperature is in 20～60 ℃ of scopes;

(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～50;

(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the compound Chinese medicinal preparation to be measured with the Radix Astragali flavone constituents, the preparation testing sample finger printing;

(6) with the finger printing of compound preparation to be measured and the contrast of above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:

I. calculate the similarity of the finger printing and the standard finger-print of compound preparation to be measured, should be 0.80～1.00;

II. in the compound preparation finger printing to be measured, non-total peak area must not surpass 10% of total peak area;

III. the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 20%.

B, the test of employing liquid chromatography are characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents:

(1) preparation of need testing solution: it is an amount of to get compound preparation to be measured, adds methanol or ethanol or water and is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Astragali and the pseudo-ginseng, comprise the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁, a kind of in the astragaloside, water or methanol or dissolve with ethanol are settled to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol: water or 0.1%～5% glacial acetic acid or 0.1%～5% formic acid or 0.005%～5% phosphoric acid solution, gradient elution, flow velocity is 0.5～2.0ml/min, detect wavelength is that one or several or evaporative light scattering detector in the 190-400nm scope detects, and column temperature is in 20～60 ℃ of scopes;

(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～40;

(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the compound preparation to be measured with Radix Astragali saponin class, arasaponin constituents, the preparation testing sample finger printing;

The method of quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng is characterized in that: this method adopts liquid chromatography test to be characterized as main finger printing and to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents:

(1) preparation of need testing solution: get compound preparation 0.1g to be measured, add the n-butyl alcohol dissolving, filter, filtrate evaporate to dryness, residue add methanol and are diluted to 5ml, as need testing solution;

(2) preparation of object of reference solution: get formononetin, with dissolve with methanol and be diluted to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile: water, gradient elution, solvent ratios is from 0 minute to 20 minutes, the ratio of acetonitrile is for to rise to 25% from 4%, from 20 minutes to 40 minutes, the ratio of acetonitrile is for to rise to 45% from 25%, from 40 minutes to 60 minutes, the ratio of acetonitrile is for rising to 70% from 45%, and from 60 minutes to 70 minutes, the ratio of acetonitrile was for to rise to 80% from 70%, from 70 minutes to 80 minutes, the ratio of acetonitrile is for rising to 100% from 80%, and flow velocity is 1ml/min, the detection wavelength is 254 ± 2nm, 25 ℃ of column temperatures;

(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;

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

II. in the compound preparation finger printing to be measured, non-total peak area must not surpass 5% of total peak area;

III. the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 10%.

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile: water, gradient elution, solvent ratios is from 0 minute to 12 minutes, the ratio of acetonitrile is 15%, and from 14 minutes to 45 minutes, the ratio of acetonitrile was for to rise to 30% from 15%, from 45 minutes to 60 minutes, the ratio of acetonitrile is for rising to 80% from 30%, and flow velocity is 1ml/min, detect wavelength 203 ± 2nm, 30 ℃ of column temperatures;

(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;

The method of quality control of the described compound preparation made from the Radix Astragali, Radix Notoginseng is characterized in that: the discrimination method of described compound preparation comprises one or more in following:

The thin layer chromatography discrimination method of the Radix Astragali in a, the compound Chinese medicinal preparation:

It is an amount of to get compound preparation to be measured, add ethyl acetate or ethanol or methanol or n-butanol extraction, filter the filtrate evaporate to dryness, residue adds the dissolving of 0.05%～5% sodium hydroxide solution, filter, filtrate is regulated pH value to 3～6 with hydrochloric acid, with ethyl acetate or n-butanol extraction, filter, filtrate evaporate to dryness, residue add ethyl acetate or methanol or dissolve with ethanol, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On lamellae or the polyamide film, with chloroform or methylene chloride-methanol or ethanol 1～30: 0.2～10 is developing solvent, launch, take out, dry, put to smoke under the rearmounted daylight or under ultra-violet lamp 365nm or the 254nm in the ammonia steam and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;

The thin layer chromatography discrimination method of astragaloside in b, the compound Chinese medicinal preparation:

It is an amount of to get compound preparation to be measured, be added on the neutral alumina post after adding methanol or dissolve with ethanol,, collect eluent with 10%～70% methanol-eluted fractions, evaporate to dryness, add after residue is dissolved in water that water-saturated n-butanol extracts and and n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On lamellae or the polyamide film, lower floor's solution with chloroform or methylene chloride-methanol or alcohol-water 1～30: 1～20: 0.2～10 is developing solvent, launch, take out, dry, spray is with 2%～50% ethanol solution of sulfuric acid, 80～160 ℃ to be heated to speckle colour developing clear, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;

The liquid chromatograph discrimination method of astragaloside in C, the compound Chinese medicinal preparation:

It is an amount of to get compound preparation to be measured, be dissolved in water the back with n-butyl alcohol or ethyl acetate extraction and and extracting solution, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol or 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, chromatographic column is that filler methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution 15%～85%: 85%～15% are mobile phase with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, detecting wavelength is that 190～410nm or evaporative light scattering detector detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Radix Notoginseng, ginsenoside Rg in d, the compound Chinese medicinal preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more thin layer chromatography discrimination method:

It is an amount of to get compound preparation to be measured, with n-butyl alcohol or ethanol or methanol or ethyl acetate or chloroform extraction, filters, and filtrate is as need testing solution; Other gets Radix Notoginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more, the preparation contrast solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adding chloroform or dichloromethane or aether backflow extracts, discard chloroform or dichloromethane or ether solution, residue adds water-saturated n-butanol or ethyl acetate extraction, extracting solution adds ammonia solution, divides and gets upper strata, evaporate to dryness, residue is with methanol or dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances, add methanol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, lower floor's solution or n-butyl alcohol ethyl acetate or the Ethyl formate-water 1～10: 0.2～2 placed below 5～40: 10～100: 5～50: 0.2～30 10 ℃ with chloroform-ethyl acetate or Ethyl formate-methanol-water: 1～15 upper strata is developing solvent, launch, take out, dry, spray is with 5～50% sulphuric acid ethanol reagent or 50% sulphate reagent, 80 ℃～160 ℃ dry by the fire to speckle colour developing clear, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the control medicinal material chromatograph, on the corresponding position of reference substance chromatograph, should show the speckle of same color, negative noiseless;

Ginsenoside Rg in e, the compound Chinese medicinal preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more liquid chromatograph differentiate:

It is an amount of to get compound preparation to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Re, Panax Notoginseng saponin R ₁In the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;

The thin layer chromatography discrimination method of one or both of formononetin in f, the compound Chinese medicinal preparation, hair stamen formononetin:

It is an amount of to get compound preparation to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Other gets in formononetin, the hair stamen formononetin reference substance one or both, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, with chloroform or methylene chloride-methanol or ethanol-ethyl acetate or Ethyl formate-water 0.5～10: 1～15: 1～20: 0.1～3 is developing solvent, launch, take out, dry, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect, in the test sample chromatograph, with reference substance chromatograph and the corresponding position of control medicinal material on, should show the same color speckle;

The liquid chromatograph discrimination method of one or both of formononetin in g, the compound Chinese medicinal preparation, hair stamen formononetin:

It is an amount of to get compound preparation to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Methanol or alcoholic solution with formononetin, hair stamen formononetin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, mobile phase A is methanol or acetonitrile, Mobile phase B is that water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, gradient elution, the detection wavelength is 190～410nm, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

The method of quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng is characterized in that: the discrimination method of described compound Chinese medicinal preparation comprises one or more in following:

It is an amount of to get compound preparation to be measured, adds ethanol extraction, filters the filtrate evaporate to dryness, residue adds the dissolving of 0.3% sodium hydroxide solution, filters, and filtrate is regulated pH value to 5～6 with hydrochloric acid, uses ethyl acetate extraction, filter, filtrate evaporate to dryness, residue add the ethyl acetate dissolving, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developing solvent with chloroform-methanol at 10: 1, launches, take out, dry, put in the ammonia steam and inspect under the smoked rearmounted ultra-violet lamp 365nm, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle;

It is an amount of to get compound preparation to be measured, be added on the neutral alumina post after adding dissolve with methanol, use 40% methanol-eluted fractions, collect eluent, evaporate to dryness, add water-saturated n-butanol after residue is dissolved in water and extract, merge n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of 13: 7: 2 of chloroform-methanol-water is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts to reach under the ultra-violet lamp 365nm daylight under and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;

It is an amount of to get compound preparation to be measured, the back that is dissolved in water water saturation n-butanol extraction and extracting solution also, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol or 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

It is an amount of to get compound preparation to be measured, uses n-butanol extraction, filters, and filtrate is as need testing solution; Other gets Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more, preparation control medicinal material solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adds the chloroform reflux, extract,, discards chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: 10 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph, the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless;

It is an amount of to get compound preparation to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Re, Panax Notoginseng saponin R ₁In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;

It is an amount of to get compound preparation to be measured, adds methanol extraction, and extracting solution is as need testing solution; Other gets in formononetin, the hair stamen formononetin one or both, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-ethyl acetate-water 2.5: 3: 4: 0.5 was developing solvent, launch, exhibition is taken out apart from 5cm, dries, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle;

It is an amount of to get compound preparation to be measured, adds methanol extraction, and extracting solution is as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

The method of quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng is characterized in that: the method for testing of described compound preparation content should comprise one or more in following:

The assay of astragaloside in a, the compound Chinese medicinal preparation:

It is an amount of to get compound preparation to be measured, be dissolved in water the back with n-butyl alcohol or ethyl acetate extraction and and extracting solution, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution 15%～85%: 85%～15% are mobile phase, detecting wavelength is that 190～410nm or evaporative light scattering detector detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains must not be less than 0.02mg;

Ginsenoside Rg in b, the compound Chinese medicinal preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more assay:

It is an amount of to get compound preparation to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, compound preparation to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount contains the ginsenoside Rg ₁Limit must not be less than 2.5mg;

(2) the per unit amount contains ginsenoside Rb ₁Limit must not be less than 3.5mg;

(3) the per unit amount contains Panax Notoginseng saponin R ₁Limit must not be less than 0.25mg;

The per unit amount contains the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The limit of summation must not be less than 6mg;

The assay of total saponins in c, the compound Chinese medicinal preparation:

It is an amount of to get compound preparation to be measured, puts in the measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured in right amount, puts in the measuring bottle, water bath method takes out immediately, and precision adds 1%～50% vanillin-glacial acetic acid solution 0.1～10ml, perchloric acid 0.1～15ml shakes up, and heats 3～50 minutes in 30～80 ℃ of water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with astragaloside or ginsenoside Rg ₁Or ginsenoside Rb ₁Or Panax Notoginseng saponin R ₁Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures trap, calculate with one point external standard method or standard curve method, compound preparation to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with astragaloside or ginsenoside Rg ₁Or ginsenoside Rb ₁Or Panax Notoginseng saponin R ₁Meter must not be less than 10mg;

The assay of one or both of formononetin in d, the compound Chinese medicinal preparation, hair stamen formononetin:

It is an amount of to get compound preparation to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Methanol or alcoholic solution with formononetin, hair stamen formononetin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, mobile phase A is methanol or acetonitrile, Mobile phase B is that water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, gradient elution, the detection wavelength is 190～410nm, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, compound preparation to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or two:

(1) the per unit amount limit that contains formononetin must not be less than 0.01mg;

(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.01mg.

The method of quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng, it is characterized in that: the method for testing of described compound preparation content is following one or more methods:

The assay of astragaloside in a, the compound Chinese medicinal preparation:

It is an amount of to get compound preparation to be measured, the back that is dissolved in water water saturation n-butanol extraction and extracting solution also, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.04mg;

It is an amount of to get compound preparation to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with one point external standard method or standard curve method, compound preparation to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount contains the ginsenoside Rg ₁Limit must not be less than 5mg;

(2) the per unit amount contains ginsenoside Rb ₁Limit must not be less than 7mg;

(3) the per unit amount contains Panax Notoginseng saponin R ₁Limit must not be less than 0.5mg;

(4) the per unit amount contains the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The limit of summation must not be less than 12mg;

The assay of total saponins in c, the compound Chinese medicinal preparation:

It is an amount of to get medicine to be measured, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision measures 0.6, puts in the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with the ginsenoside Rg ₁Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, compound preparation to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg ₁Meter must not be less than 20mg;

It is an amount of to get compound preparation to be measured, adds methanol extraction, and extracting solution is as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, compound preparation to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or two:

(1) the per unit amount limit that contains formononetin must not be less than 0.02mg;

(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.02mg.

The method of quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng, it is characterized in that: the assay result of described traditional medicine Injectio, calculate astragaloside, formononetin, calycosin, total saponins, Panax Notoginseng saponin R according to percentage by weight ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁In the total content of all or part of material account for deduction adjuvant and outer more than 25% of total solid of moisture in the preparation.

Compared with prior art, the present invention's quality of the compound Chinese medicinal preparation made with the Radix Astragali, Radix Notoginseng of perfect control more.The Chinese medicine ingredients complexity, if only with wherein one, two kind of composition illustrate its inherent quality, has certain one-sidedness, more can't judge the index components of its drug effect.Therefore the applicant has formulated with the Radix Astragali flavone constituents and has been characterized as main finger printing and is characterized as the quality that main finger printing, discriminating and assay are controlled compound Chinese medicinal preparation comprehensively with Radix Astragali saponin class, arasaponin constituents.But because contained complex chemical composition between each medical material in the compound Chinese medicinal preparation, formulation, discriminating and assay to finger printing cause interference, cause discriminating, assay and each several part finger printing feature instability, so must control mobile phase, developing solvent isochromatic spectrum condition, just can obtain good thin layer chromatography, contain survey condition and finger printing.That is to say, because each composition interference effect each other in the prescription, cause the finger printing characteristic peak of the Radix Astragali in the compound Chinese medicinal preparation, Radix Notoginseng part to change, and have only the condition of the present invention of employing, just can obtain ideal thin layer chromatography, contain survey condition and finger printing.

Proof by experiment, method of quality control of the present invention is more effective to the quality control of the compound Chinese medicinal preparation product made with the Radix Astragali, Radix Notoginseng, and method precision, stability are all higher.

Experimental example 1 is characterized as main finger printing with the Radix Astragali flavone constituents

A, experimental apparatus, reagent and sample:

Reference substance: formononetin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute

B, chromatographic condition and system suitability experiment:

1. the selection of chromatographic column:

In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, has tried out Inertsil ODS-3 (250mm * 4.6mm, 5 μ m) Kromasil C respectively ₁₈(200mm * 4.6mm, 5 μ m), Diamonsil C ₁₈The chromatographic column of (250mm * 4.6mm, 5 μ m) three kinds of trades mark, the result shows that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, wherein Diamonsil C ₁₈The chromatographic column separating effect is best, and post is imitated the highest (calculating with the object of reference formononetin).So finally select Diamonsil C for use ₁₈(250mm * 4.6mm, 5 μ m) are the experimentation post.

2. the selection of mobile phase:

Investigated (1) methanol-0.05mol/L sodium dihydrogen phosphate (20: 80) in the research process respectively, (2) acetonitrile-0.05mol/L sodium dihydrogen phosphate (10: 90), (3) acetonitrile-0.05mol/L sodium hydrogen phosphate (gradient elution) (4) acetonitrile-water (gradient elution), solvent ratios is from 0 minute to 20 minutes, the ratio of acetonitrile is for to rise to 25% from 4%, from 20 minutes to 40 minutes, the ratio of acetonitrile is for to rise to 45% from 25%, from 40 minutes to 60 minutes, the ratio of acetonitrile is for rising to 70% from 45%, and from 60 minutes to 70 minutes, the ratio of acetonitrile was for to rise to 80% from 70%, from 70 minutes to 80 minutes, the ratio of acetonitrile was for to rise to 100% from 80%.The result shows that peak shape is relatively poor under mobile phase (1) condition, and it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (2) the mobile phase condition, peak shape is relatively poor, separates bad; (3) under the condition, the peak hangover is serious, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.

3. detection wavelength determination:

In the research under acetonitrile-water (gradient elution) mobile phase condition, investigated the chromatographic peak situation under different-waveband typical wavelengths 230,254,270,300 respectively, the result shows, 254nm can take into account kurtosis, separating degree and the baseline of each composition chromatographic peak when detecting, so select 254nm to detect wavelength for this product finger printing.

4. instrument, chromatographic column and integral parameter:

4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved LC-10Avp high performance liquid chromatograph for use, the WML-2010 chromatographic work station.Chromatographic column is Diamonsil C ₁₈(250mm * 4.6mm, 5 μ m); 40 ℃ of column temperatures, flow velocity 1.0ml/min.

4.2 integral parameter: peak width: 10; Slope: 100; Smallest peaks area: 500.So setting can be avoided some very calculating of the chromatographic peak of small size (unimodal area accounts for total peak area less than 0.5%), guarantees the dependency with object of reference simultaneously.

5. the preparation of need testing solution:

Get compound Chinese medicinal preparation 0.1g to be measured, add the n-butyl alcohol dissolving, filter, filtrate evaporate to dryness, residue add methanol and are diluted to 5ml, as need testing solution.

6. the preparation of object of reference solution:

Get formononetin, add methanol and make the solution that every 1ml contains 0.1mg, as object of reference solution.

7. finger printing and technical parameter:

Formulate standard finger-print according to 10 batch samples, test sample finger printing and standard finger-print are compared, similarity is all between 0.90～1.00.

Experimental example 2 is characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents

A, experimental apparatus, reagent and sample:

Reference substance: ginsenoside Rg ₁: Nat'l Pharmaceutical ﹠ Biological Products Control Institute

B, chromatographic condition and system suitability experiment:

1. the selection of chromatographic column:

In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, tried out Zorbax, Inertsil ODS-3 respectively, Diamonsil ODS (is C18,4.6mm * 200mm, 5 μ m) chromatographic column of three kinds of trades mark, result show that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, and wherein Diamonsil ODS chromatographic column separating effect is best, post is imitated the highest, can reach 5400 (calculating with the object of reference ginsenoside Rg1).So finally selecting Diamonsil ODS chromatographic column (4.6mm * 200mm, 5 μ m) for use is the experimentation post.

2. the selection of mobile phase:

Investigated (1) methanol-water (20: 80) in the research process respectively, (2) acetonitrile-water (10: 90), (3) (the gradient elution volume proportion is from 0 minute to 12 minutes to acetonitrile-0.05mol/L sodium dihydrogen phosphate (gradient elution) (4) acetonitrile-water (gradient elution), the ratio of acetonitrile is 15%, from 14 minutes to 45 minutes, the ratio of acetonitrile is for rising to 30% from 15%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was for rising to 80% from 30%) four kinds of flow phase system.The result shows that peak shape is relatively poor under mobile phase (1) condition, and it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (2) the mobile phase condition, peak shape is relatively poor, separates bad; (3) under the condition, the peak hangover is serious, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.

3. detection wavelength determination:

In the research under acetonitrile-water (gradient elution) mobile phase condition, investigated the chromatographic peak situation under different-waveband typical wavelengths 203,210,230,254 respectively, the result shows that chromatographic peak is more under 203nm, peak shape is better, so finally select for use 203nm ± 2 as detecting wavelength.

4. instrument, chromatographic column and integral parameter:

4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved Agilent1100 series of high efficiency chromatograph of liquid for use, the Chemstation chromatographic work station.Chromatographic column is Diamonsil ODS (4.6mm * 200mm, 5 μ m); 40 ℃ of column temperatures, flow velocity 1.0ml/min.

4.2 integral parameter: Slope Sensitivity:1, peak width:0.05, smallest peaks area are 5% of object of reference (S) peak-to-peak area, minimum peak height be the S peak-to-peak high 5%.So setting can be avoided some very calculating of the chromatographic peak of small size (unimodal area accounts for total peak area less than 0.5%), guarantees the dependency with object of reference simultaneously.

5. the preparation of need testing solution:

It is an amount of that precision takes by weighing this product, adds water and make the solution that every 1ml contains 50mg, promptly.

6. the preparation of object of reference solution:

The ginsenoside Rg ₁Be one of Radix Notoginseng main active, its integral area proportion in finger printing more greatly and more stable is taken into account the research of intermediate and medical material simultaneously, therefore selected ginsenoside Rg ₁As object of reference.

7. finger printing and technical parameter:

The thin layer chromatography discrimination method of the Radix Astragali in experimental example 3 compound Chinese medicinal preparation:

Feature for the outstanding Radix Astragali, selected Radix Astragali control medicinal material as its feature speckle, but owing to there is composition like more, the polar phase close in the compound Chinese medicinal preparation with Radix Astragali control medicinal material structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition launches Radix Astragali control medicinal material:

Condition	Problem
Condition	Problem	N-butyl alcohol-glacial acetic acid-water (6: 4: 3) silica gel H lamellae acetone-glacial acetic acid-water (8: 2: 1) silica gel g thin-layer plate methylene chloride-methanol (7-3) silica gel G F ₂₅₄Lamellae chloroform-acetone-formic acid, (20: 2: 1) silica gel g thin-layer plate chloroform-acetone-methanol, (20: 3: 2) silica gel H lamellae chloroform-acetone-methanol, (20: 2: 10) silica gel g thin-layer plate chloroform-methanol, (10-1) silica gel g thin-layer plate	It is clear that feminine gender has the unintelligible feminine gender of interference characteristic spot to have the feminine gender of interference to have the feminine gender of interference to have the reference substance of interference to be expanded to the forward position separation, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with chloroform-methanol (10-1), with this understanding, the Rf value of Radix Astragali control medicinal material feature speckle is moderate, and it is clear to separate with other speckle, negative noiseless.

The thin layer chromatography discrimination method of astragaloside in experimental example 4 compound Chinese medicinal preparation:

Feature for the outstanding Radix Astragali, selected astragaloside as its feature speckle, but owing to there is composition like more, the polar phase close in the compound Chinese medicinal preparation with the astragaloside structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition launches astragaloside:

Condition	Problem
Condition	Problem	Chloroform-acetone-water (10-8-0.5) silica gel g thin-layer plate dichloromethane-acetone-water (10-8-0.5) silica gel g thin-layer plate dichloromethane-acetone (2-9) silica gel H lamellae chloroform-acetone (10-5) silica gel G F ₂₅₄Lamellae chloroform-ethanol (13-7) silica gel H lamellae chloroform-methanol (13-7) silica gel g thin-layer plate chloroform-methanol-water (13-7-2) lower floor solution	Feminine gender has the feminine gender of interference to have the reference substance of interference exhibition to forward position feminine gender to have the feminine gender of interference to have the feminine gender of interference to have interference separation clear, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with lower floor's solution of chloroform-methanol-water (13-7-2), with this understanding, the Rf value of astragaloside feature speckle is moderate, and it is clear to separate with other speckle, and feminine gender is noiseless.

The liquid chromatograph discrimination method of astragaloside in experimental example 5 compound Chinese medicinal preparation:

Feature for the outstanding Radix Astragali, except the thin layer discrimination method, selected astragaloside as its characteristic component, but owing to there is composition like more, the polar phase close in the compound Chinese medicinal preparation with the astragaloside structure, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase astragaloside are separated:

Condition	Problem
Condition	Problem	Methyl alcohol-0.02mol/L sodium hydrogen phosphate (70: 30) eight alkyl silane bonded silica gel acetonitriles-0.02mol/L sodium hydrogen phosphate (50: 50) octadecylsilane chemically bonded silica methyl alcohol-0.05mol/L sodium hydrogen phosphate (40: 60) octadecylsilane chemically bonded silica acetonitrile-0.05mol/L sodium hydrogen phosphate (30: 70) octadecylsilane chemically bonded silica methyl alcohol-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane chemically bonded silica acetonitrile-methyl alcohol-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane chemically bonded silica acetonitrile-water (32: 68) octadecylsilane chemically bonded silica	The too fast feminine gender of the too fast appearance time of appearance time has the slightly asymmetric feminine gender of the peak shape of interference to have the slightly asymmetric retention time of the peak shape of interference moderate; The peak is capable sharp-pointed; Symmetry, negative noiseless

Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water (32: 68) is a mobile phase, and with this understanding, the astragaloside retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless.

Experimental example 6 compound Chinese medicinal preparation ginsenoside Rgs ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The thin layer chromatography discrimination method

For the feature of outstanding Radix Notoginseng, selected the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁As its feature speckle, but owing to exist more and the ginsenoside Rg in the medical material ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Structure is close, composition like the polar phase, and usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:

Condition	Problem
Condition	Problem	(15: 40: 22: (10: 40: 15: lower floor's solution silica gel g thin-layer plate n-butanol-Ethyl formate of 5) placing below 10 ℃-methyl alcohol (5-1-5) silica gel g thin-layer plate n-butanol-Ethyl formate-ethanol (2-1.5-0.5) silica gel H lamellae was determined condition to lower floor's solution silica gel g thin-layer plate chloroform-ethyl acetate of 10) placing below 10 ℃-formic acid-water to lower floor's solution silica gel g thin-layer plate n-butanol-Ethyl formate that chloroform-Ethyl formate-water (20: 60: 10) is placed below 10 ℃-methyl alcohol (10-1-5) silica gel H lamellae chloroform-Ethyl formate-formic acid-water: chloroform-ethyl acetate-methanol-water (15: 40: 22: lower floor's solution silica gel g thin-layer plate of 10) placing below 10 ℃	Reference substance is expanded to the forward position reference substance and is expanded to the forward position reference substance and does not separate, feminine gender has the reference substance of interference not separate, feminine gender has the reference substance of interference not separate, feminine gender has the reference substance of interference not separate, feminine gender has interference separation clear, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the silica gel g thin-layer plate to be immobile phase, with chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developing solvent, with this understanding, the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Rf value moderate, it is clear to separate with other speckle, negative noiseless.

Ginsenoside Rg in experimental example 7 compound Chinese medicinal preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The liquid chromatograph discrimination method

For the feature of outstanding Radix Notoginseng, selected the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁As its characteristic component, but owing to exist more and the ginsenoside Rg in the medical material ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Structure is close, composition like the polar phase, and usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and mobile phase to the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Separate:

Condition	Problem
Condition	Problem	Methanol-0.05mol/L sodium dihydrogen phosphate (70: 30) octadecylsilane chemically bonded silica acetonitrile-0.05mol/L sodium dihydrogen phosphate (55: 45) octadecylsilane chemically bonded silica methanol-0.05mol/L sodium hydrogen phosphate (80: 20) eight alkyl silane bonded silica gel methanol-0.05mol/L sodium hydrogen phosphate (80: 20) octadecylsilane chemically bonded silica acetonitrile-0.02mol/L potassium dihydrogen phosphate (90: 10) eight alkyl silane bonded silica gel methanol-0.02mol/L potassium dihydrogen phosphate (85: 15) octadecylsilane chemically bonded silica acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, from 15 minutes to 21 minutes, the ratio of acetonitrile rises to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20 % octadecylsilane chemically bonded silicas	The too fast feminine gender of the too fast appearance time of appearance time has the feminine gender of interference to have the feminine gender of interference to have the feminine gender of interference to have the retention time of interference moderate; The peak is capable sharp-pointed; Symmetry, negative noiseless

Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile is 20%, with this understanding, and the people ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless.

Experimental example 8 Astragaloside contents are measured

Instrument and reagent

(1) key instrument:

High performance liquid chromatograph P-426 Alltech

Evaporative light scattering detector 2000ES Alltech

High performance liquid chromatograph 1100 series Agilent

Ultrasonic washing unit KQ250B Kunshan Ultrasonic Instruments Co., Ltd.

Emerging great achievement instrument company in electric vacunm drying case ZK-30ABX Beijing

Electronic analytical balance AE240 Mettler

(2) reagent: astragaloside: Nat'l Pharmaceutical ﹠ Biological Products Control Institute

Used methanol, acetonitrile are chromatographically pure, and all purchasing in Tian Jinsi friend biomedical technology development company water is redistilled water, and other reagent is analytical pure.

Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).

1 chromatographic condition

Chromatographic column: Platihum C ₁₈4.6 * 250mm 5 μ m;

Mobile phase: acetonitrile-water (32: 68);

Flow velocity: 1.0ml/min;

The evaporation photodetector detects: drift tube temperature: 110 ℃, and throughput: 2.7L/min;

Column temperature: 30 ℃.

Obtain astragaloside, test sample, negative chromatogram according to above-mentioned condition; The astragaloside peak reaches baseline separation, separates with close peak that clear fully retention time is moderate, and peak shape is sharp-pointed, symmetry, and number of theoretical plate calculates by the astragaloside peak should be not less than 2000.

The preparation of 2 reference substance solution is accurate respectively to take by weighing through 24 hours astragaloside reference substance of phosphorus pentoxide desiccator drying under reduced pressure in right amount, adds methanol and makes the mixed solution that every 1ml contains 0.2mg, promptly.

This product under the content uniformity item is got in the preparation of 3 need testing solutions, get content, porphyrize, therefrom precision takes by weighing about 5g, add water 20ml dissolving back water saturation n-butanol extraction, 40ml and also n-butyl alcohol liquid at every turn use ammonia solution thorough washing 2 times, each 40ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue add water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml successively, 40% ethanol 30ml, 70% ethanol 80ml eluting is collected 70% ethanol elution part, evaporate to dryness, residue dissolves with methanol 5ml, shake up, filter, get subsequent filtrate promptly.

4 algoscopys precision are respectively drawn reference substance solution 10 μ l, 20 μ l, and need testing solution 200 μ l inject chromatograph of liquid, measure, with the content of external standard two-point method logarithmic equation calculating astragaloside, promptly.

5 linear relationships are investigated accurate astragaloside (0.2085mg/ml) reference substance solution 2,4,8,12, the 16 μ l that draw, inject chromatograph of liquid, common logarithm value with the amount (μ g) of astragaloside is an abscissa, and the common logarithm value of peak area is that vertical coordinate is figure, the drawing standard curve.

The astragaloside linear relationship

Numbering	Astragaloside (μ g)	Astragaloside common logarithm value	Peak area	Peak area common logarithm value
Numbering	Astragaloside (μ g)	Astragaloside common logarithm value	Peak area	Peak area common logarithm value	1 2 3 4 5	0.417 0.834 1.668 2.502 3.336	-0.3799 -0.07883 0.2222 0.3983 0.5232	461895 971004 2022680 32111269 4312244	5.6645 5.9872 6.3059 6.4929 6.6347

Astragaloside regression equation: Y=1.0700X+6.0705;

Astragaloside correlation coefficient: γ=0.9999;

Astragaloside is good in 0.417～3.336 μ g scope internal linear.

Accurate reference substance solution (0.1668mg/ml) the 10 μ l that draw of 6 precision test inject chromatograph of liquid, continuous sample introduction 5 times.

The test of reference substance solution precision

Test number (TN)	1	2	3	4	5	Average	RSD(％)
Test number (TN)	1	2	3	4	5	Average	RSD(％)	Peak area	2031539	2041338	2021467	2015966	2030855	2028233	0.48

The result shows that precision is good.

7 stability tests

7.1 accurate reference substance solution (0.1668mg/ml) the 10 μ l that draw of reference substance stability test inject chromatograph of liquid, inject chromatograph of liquid, respectively at 0,6,12,24,48 hour replication 1 time, and record peak area integrated value.

The reference substance solution stability test

Time (h)	0	6	12	24	48	Average	RSD(％)
Time (h)	0	6	12	24	48	Average	RSD(％)	Peak area	2031539	2041338	2021467	2015966	2030855	2028233	0.48

The result shows that reference substance solution is good at 48 hours internal stabilities.

Draw same need testing solution 20 μ l 7.2 the test sample stability test is accurate, inject chromatograph of liquid, respectively 0,6,12,24,48 hour replication 1 time, record peak area integrated value.

The need testing solution stability test

Time (h)	0	6	12	24	48	Average	RSD(％)
Time (h)	0	6	12	24	48	Average	RSD(％)	Content (mg/ bottle)	0.0264	0.0259	0.0263	0.0261	0.0266	0.0263	1.03

The result shows that need testing solution is good at 48 hours internal stabilities.

8 replica tests are got this product under the content uniformity item, get content, porphyrize, therefrom precision takes by weighing about 5g (totally 5 parts), handles by the preparation of text need testing solution and the method for measuring under the item, the therefrom accurate respectively 20 μ l that draw, inject chromatograph of liquid, measure, promptly.

Test sample replica test result

Numbering	1	2	3	4	5	Average	RSD(％)
Numbering	1	2	3	4	5	Average	RSD(％)	Content (mg/ bottle)	0.0267	0.0281	0.0274	0.0277	0.0273	0.0274	1.89

The result shows that repeatability is good.

The application of sample absorption method is adopted in the test of 9 average recoveries, gets this product under the content uniformity item, gets content, porphyrize, and therefrom precision takes by weighing about 2.5g (totally 6 parts), splits in the tool plug conical flask; Accurate astragaloside reference substance aqueous solution (0.1233mg/ml) 1ml (totally 6 parts) that draws, split in the above-mentioned tool plug conical flask, add water 20ml dissolving back water saturation n-butanol extraction, 40ml and also n-butyl alcohol liquid at every turn use ammonia solution thorough washing 2 times, each 40ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue add water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml successively, 40% ethanol 30ml, 70% ethanol 80ml eluting is collected 70% ethanol elution part, evaporate to dryness, residue dissolves with methanol 5ml, shake up, filter, get subsequent filtrate promptly.The accurate subsequent filtrate 20 μ l that draw inject chromatograph of liquid, measure, promptly.

The content of astragaloside is respectively 0.05448mg/g in the known after measured compound Chinese medicinal preparation.

The test of astragaloside average recovery

Numbering	Test sample weighing (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5 6	2.51143 2.50085 2.50272 2.50188 2.50392 2.50339	0.1368 0.1362 0.1363 0.1363 0.1364 0.1364	0.1233 0.1233 0.1233 0.1233 0.1233 0.1233	0.2587 0.2570 0.2556 0.2573 0.2585 0.2567	98.83 97.94 96.69 98.12 99.03 97.54

Astragaloside average recovery rate=98.03%; RSD=0.88%

Test agent was three batches during 9 three batch sample Astragaloside contents were measured and got, and pressed the described method of text and handled, and the accurate 20 μ l sample introductions of drawing are measured.

Test agent Astragaloside content measurement result in three batches

Lot number	Astragaloside content (mg/ bottle)
Lot number	Astragaloside content (mg/ bottle)	1 batch 2 batches 3 batches	0.0302 0.0285 0.0294

Experimental example 9 total saponin contents are measured

1 instrument, reagent

1.1 instrument: the general logical TU-1810SPC ultraviolet/visible spectrophotometer of analysing

SARTORIUS BP211D electronic analytical balance

1.2 reagent: vanillin analytical pure Tianjin recovery fine chemistry industry institute

Methanol chromatographically pure J.T.Baker

Glacial acetic acid analytical pure Shanghai reagent one factory

Chemical plant, prosperous source, perchloric acid analytical pure Tianjin

The pure water WAHAHA

2 methods and result

Take by weighing the ginsenoside Rg 2.1 detect the selection precision of wavelength ₁5.14mg, put in the 100ml measuring bottle, add an amount of methanol supersound process (power 250W, frequency 33KHz) and make dissolving, add methanol to scale, shake up, precision is measured 1.2ml, puts in the 10ml tool plug test tube water bath method solvent, take out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml shakes up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds, shake up, scan in 700～400nm wave-length coverage, the result shows that astragaloside has absorption maximum at the 547nm place, blank noiseless, therefore selecting 547nm is the detection wavelength of total saponins in the spectrophotometry compound Chinese medicinal preparation.

2.2 the preparation ginsenoside Rg of reference substance solution ₁5mg adds methanol and makes the solution that every 1ml contains 0.05mg, promptly.

2.3 this product under the content uniformity item is got in the preparation of need testing solution, gets content, mixing, therefrom get about 50mg, the accurate title, decide, and puts in the 25ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHZ) and make dissolving, take out, put to room temperature, add methanol to scale, shake up, precision is measured 1.0ml, put in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), measure trap at the wavelength place of 547nm.Calculate with one point external standard method, promptly.

The investigation precision of 3 linear relationships takes by weighing the ginsenoside Rg ₁Reference substance 5.03mg puts in the 100ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHZ) makes dissolving, take out, put to room temperature, add methanol to scale, shake up, precision measures 0.4,0.8,1.2,1.6,2.0ml, split in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), measure trap at the wavelength place of 547nm.With the trap is vertical coordinate, and the amount of astragaloside (μ g) is an abscissa, the drawing standard curve.

Regression equation Y=0.0062X+0.0047; R=0.9999

The ginsenoside Rg ₁Linear good in 20.12～100.6 μ g scopes.

The ginsenoside Rg ₁The standard curve determination data

Numbering	The ginsenoside Rg ₁(μg)	Trap
Numbering	The ginsenoside Rg ₁(μg)	Trap	1 2 3 4 5	20.12 40.24 60.36 80.48 100.6	0.127 0.255 0.377 0.502 0.624

3 precision test precision is measured reference substance solution (0.0503mg/ml) 1.2ml, puts in the 10ml tool plug test tube water bath method solvent, take out accurate 5% vanillin-glacial acetic acid solution 0.2ml, the perchloric acid 0.8ml of adding, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, press operation under the text algoscopy item, METHOD FOR CONTINUOUS DETERMINATION 5 times.

The precision experiment

Numbering	1	2	3	4	5	X is average	RSD％
Numbering	1	2	3	4	5	X is average	RSD％	Absorbance	0.376	0.374	0.378	0.377	0.379	0.377	0.51

5 stability tests

5.1 the stability test precision of reference substance solution is measured reference substance solution (0.0503mg/ml) 1.2ml, puts in the 10ml tool plug test tube water bath method solvent, take out accurate 5% vanillin-glacial acetic acid solution 0.2ml, the perchloric acid 0.8ml of adding, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, press operation under the text algoscopy item, in the time of 0,10,20,40,60 minute, measure respectively.

The reference substance solution stability experiment

Time (min)	0	10	20	40	60	Average	RSD％
Time (min)	0	10	20	40	60	Average	RSD％	Absorbance	0.375	0.368	0.381	0.374	0.372	0.374	1.27

5.2 the stability experiment of need testing solution is got this product under this product content uniformity item, gets content, porphyrize is therefrom got about 50mg, by operating under the text algoscopy item, measures in the time of 0,10,20,40,60 minute respectively.

The need testing solution stability experiment

Time (min)	0	10	20	40	60	Average	RSD％
Time (min)	0	10	20	40	60	Average	RSD％	Content (mg/ bottle)	12.33	12.35	12.29	12.19	12.05	12.24	1.01

6 replica tests are got this product under this product content uniformity item, get content, and porphyrize is therefrom got about 50mg (totally 5 parts), by operating under the text algoscopy item.

Repeated experiment

Numbering	1	2	3	4	5	X is average	RSD％
Numbering	1	2	3	4	5	X is average	RSD％	Content (mg/ bottle)	12.12	12.26	12.35	12.41	12.34	12.30	0.92

7 recovery tests adopt the application of sample absorption method, get this product under the content uniformity item, get content, and porphyrize is therefrom got about 25mg (totally 5 parts), and accurate the title decides, and splits in the 25ml measuring bottle; Precision is measured the ginsenoside Rg ₁Reference substance solution (0.624mg/ml) 1ml (totally 5 parts), the accurate title, decide, and splits in the above-mentioned 25ml measuring bottle, adds water and make dissolving and fixed to scale in right amount, shake up, precision is measured 1ml, puts in the 10ml tool plug test tube, by operating under the text algoscopy item, with the retinue solvent is blank, measure trap in accordance with the law, press one point external standard method and calculate, promptly.

The content of total saponins: 23.55mg/g in the compound Chinese medicinal preparation

The average recovery experiment

Numbering	Test sample weighing (mg)	Total saponins amount (mg) in the test sample	The ginsenoside Rg ₁Addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (mg)	Total saponins amount (mg) in the test sample	The ginsenoside Rg ₁Addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	23.35 25.12 25.08 24.47 24.88	0.5506 0.5923 0.5914 0.5770 0.5867	0.624 0.624 0.624 0.624 0.624	1.1574 1.2090 1.2093 1.1882 1.2032	97.24 98.83 99.02 97.95 98.81

Average recovery rate=98.37% RSD=0.77%

8 three batches of pilot scale sample sizes are measured

Get three batches in this product sample, press the described method of text and handle.

Three batch sample assay results

Lot number	Total saponins (mg/ bottle)
Lot number	Total saponins (mg/ bottle)	1 2 3	14.07 12.26 13.48

Experimental example 10 Panax Notoginseng saponin Rs ₁, the ginsenoside Rg ₁, ginsenoside Rb ₁Assay

1 instrument and reagent

1.1 instrument:

Alltech UVIS-201 high performance liquid chromatograph, Alltech HPLC system work station (U.S.)

KQ250B ultrasonic washing unit (Kunshan Ultrasonic Instruments Co., Ltd.)

ZK-30ABX electric vacunm drying case (in Beijing emerging great achievement instrument company)

Mettler AE240 electronic balance (Shanghai Mei Tele company)

1.2 reagent:

Panax Notoginseng saponin R ₁: Nat'l Pharmaceutical ﹠ Biological Products Control Institute

The ginsenoside Rg ₁: Nat'l Pharmaceutical ﹠ Biological Products Control Institute

Ginsenoside Rb ₁: Nat'l Pharmaceutical ﹠ Biological Products Control Institute

Used methanol, acetonitrile are chromatographically pure, all purchase the company in German Merck

Water is redistilled water, and other reagent is analytical pure.

2 chromatographic conditions

Chromatographic column: Diamonsil (TM) C ₁₈, 5 μ m; 250 * 4.6mm;

Mobile phase: acetonitrile-water is a mobile phase, gradient elution;

Gradient elution system

Time (min)	Acetonitrile (%)	Water (%)
Time (min)	Acetonitrile (%)	Water (%)	0 15 21 25	20 40 20 20	80 60 80 80

Flow velocity: 1ml/min, twice sampling interval equilibration time: 3min;

Detect wavelength: 203nm;

Column temperature: 30 ℃;

Sample size: 10 μ l

Number of theoretical plate calculates by the ginsenoside Rg1 peak should be not less than 3000.

Detect the selection Panax Notoginseng saponin R of wavelength ₁, the ginsenoside Rg ₁, ginsenoside Rb ₁, all absorption maximum is arranged at 203nm wavelength place, therefore select the detection wavelength of 203nm for use as this product.

Panax Notoginseng saponin R is got in the preparation of 3 reference substance solution ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁Reference substance is an amount of, accurate claims surely, adds methanol respectively and makes every ml and contain Panax Notoginseng saponin R ₁0.62mg, the ginsenoside Rg ₁1.42mg and ginsenoside Rb ₁2.14mg solution, as stock solution; Draw each 1ml of above-mentioned solution more respectively, put in the same 5ml measuring bottle, add methanol, shake up, promptly get every ml and contain Panax Notoginseng saponin R to scale ₁0.124mg, the ginsenoside Rg ₁0.284mg and ginsenoside Rb ₁0.428mg reference substance solution.

This product under the content uniformity item is got in the preparation of 4 need testing solutions, gets content, mixing, therefrom precision takes by weighing 2.5g, puts in the 25ml measuring bottle, adds methanol 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, take out, put to room temperature, add methanol to scale, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m).

Preparation photograph this product prescription of 5 negative need testing solutions takes by weighing other composition except that Radix Notoginseng total arasaponins, makes negative test sample by this product preparation technology.Precision takes by weighing 0.31428g, according to the preparation below method preparation of need testing solution, as negative need testing solution.

The above-mentioned chromatographic condition of 6 system suitability experimental evidences obtains Panax Notoginseng saponin R ₁, the ginsenoside Rg ₁, ginsenoside Rb ₁, mix reference substance, sample, negative chromatogram, number of theoretical plate n is greater than 3000, in the sample each reference substance peak separate with close peak clear fully, separating degree is greater than 1.5, and is negative noiseless.

Panax Notoginseng saponin R is got in the investigation of 7 linear relationships ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁Reference substance is an amount of, accurate claims surely, adds methanol respectively and makes every ml and contain Panax Notoginseng saponin R ₁0.62mg, the ginsenoside Rg ₁1.42mg and ginsenoside Rb ₁2.14mg solution, as stock solution; Precision measure aforementioned reference substance stock solution each 0.2,0.4,0.8,1.2,1.6,2.0ml, split in the 5ml measuring bottle, add methanol to scale, shake up, the therefrom accurate respectively 10 μ l that draw, inject chromatograph of liquid successively, with the peak area is vertical coordinate, and sample size (μ g) is figure for abscissa, the drawing standard curve.

Regression equation is: Panax Notoginseng saponin R ₁: Y=208361.76X-1972.29 r=0.9995;

The ginsenoside Rg ₁: Y=293933.01X-13982.75 r=0.9994;

Ginsenoside Rb ₁: Y=192503.18X-9202.56 r=0.9993.

The result shows: Panax Notoginseng saponin R ₁Good in 0.248-2.480 μ g scope internal linear relation;

The ginsenoside Rg ₁Good in 0.568-5.680 μ g scope internal linear relation;

Ginsenoside Rb ₁Good in 0.856-8.560 μ g scope internal linear relation.

Linear relationship test determination result

Panax Notoginseng saponin R ₁X (μ g) peak area	0.248 50263	0.496 100526	0.992 200106	1.488 305920	1.984 413269	2.480 511633
Panax Notoginseng saponin R ₁X (μ g) peak area	0.248 50263	0.496 100526	0.992 200106	1.488 305920	1.984 413269	2.480 511633	The ginsenoside Rg ₁X (μ g) peak area	0.568 160211	1.136 319838	2.272 652866	3.408 984424	4.544 1298968	5.680 1675375
Ginsenoside Rb ₁X (μ g) peak area	0.856 160162	1.712 338350	3.424 629600	5.136 961552	6.848 1313361	8.560 1650024	The ginsenoside Rg ₁X (μ g) peak area	0.568 160211	1.136 319838	2.272 652866	3.408 984424	4.544 1298968	5.680 1675375

Reference substance mixed solution (Panax Notoginseng saponin R is got in the test of 8 precision ₁0.124mg/ml, the ginsenoside Rg ₁0.284mg/ml and ginsenoside Rb ₁0.428mg/ml), METHOD FOR CONTINUOUS DETERMINATION 5 times is investigated reference substance solution precision.

The precision test

Test number (TN)	1	2	3	4	5	Average	RSD(％)
Test number (TN)	1	2	3	4	5	Average	RSD(％)	Panax Notoginseng saponin R ₁The peak area ginsenoside Rg ₁Peak area ginsenoside Rb ₁Peak area	246053 742870 799153	251946 744876 790713	245960 746459 774498	249961 741460 797000	248515 737323 784495	248487 742598 789172	1.03 0.47 1.27

9 stability tests

9.1 the reference substance solution stability test is got reference substance mixed solution (Panax Notoginseng saponin R ₁0.124mg/ml, the ginsenoside Rg ₁0.284mg/ml and ginsenoside Rb ₁0.428mg/ml), measure at 0,2,6,10,24 hour sample introduction respectively, investigate reference substance solution stability.

The stability test of reference substance solution

Time (h)	0	2	6	10	24	Average	RSD(％)
Time (h)	0	2	6	10	24	Average	RSD(％)	Panax Notoginseng saponin R ₁The peak area ginsenoside Rg ₁Peak area ginsenoside Rb ₁Peak area	251645 751539 791325	250980 744648 794487	249983 752846 793693	256897 760121 787720	255921 745537 789215	253085 750938 791288	1.22 0.83 0.36

9.2 the stability test of need testing solution is got this product under the content uniformity item, gets content, mixing, and therefrom precision takes by weighing 2.50824g, by operating under the preparation of text need testing solution, measures at 0,2,6,10,24 hour sample introduction respectively.

Stability test

Time (h)	0	2	6	10	24	Average	RSD(％)
Time (h)	0	2	6	10	24	Average	RSD(％)	Panax Notoginseng saponin R ₁(mg/ bottle) ginsenoside Rg ₁(mg/ bottle) ginsenoside Rb ₁(mg/ bottle)	0.6235 3.015 4.225	0.6184 3.104 4.318	0.6312 3.027 4.264	0.6159 3.083 4.305	0.6201 3.052 4.221	0.6218 3.056 4.267	0.95 1.22 1.04

The result shows: need testing solution is basicly stable in 24 hours.

10 replica tests are got this product under the content uniformity item, get content, and mixing is therefrom got about 2.5g (totally 5 parts), by operating under preparation of text algoscopy need testing solution and the mensuration item.

Replica test

Numbering	1	2	3	4	5	Average	RSD(％)
Numbering	1	2	3	4	5	Average	RSD(％)	Panax Notoginseng saponin R ₁(mg/ bottle) ginsenoside Rg ₁(mg/ bottle) ginsenoside Rb ₁(mg/ bottle)	0.6188 3.112 4.307	0.6254 3.087 4.314	0.6195 3.049 4.295	0.6207 3.055 4.303	0.6224 3.018 4.281	0.6214 3.064 4.300	0.42 1.18 0.29

The result shows that this law repeatability better.

11, the application of sample absorption method is adopted in average recovery test, gets this product under the content uniformity item, gets content, and mixing is therefrom got about 1.25g (totally 5 parts), puts respectively in the 25ml measuring bottle, and precision is measured mixing reference substance solution 4ml (Panax Notoginseng saponin R wherein in addition ₁, the ginsenoside Rg ₁And ginsenoside Rb ₁Concentration be: 0.399mg/ml, 1.751mg/ml, 2.432mg/ml), add an amount of supersound process of methanol (power 250W, frequency 33KHz) and make dissolving, take out, put to room temperature, add methanol to scale, shake up, filter with microporous filter membrane (0.45 μ m), the accurate 10 μ l that draw, inject chromatograph of liquid, measure, promptly.

Panax Notoginseng saponin R in the known after measured compound Chinese medicinal preparation ₁, the ginsenoside Rg ₁And ginsenoside Rb ₁Content be 1.190,5.865 respectively, 8.232mg/g.

Panax Notoginseng saponin R ₁The average recovery test

Numbering	Test sample weighing (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	1.25042 1.24957 1.25038 1.24852 1.25662	1.488 1.487 1.488 1.486 1.495	1.596 1.596 1.596 1.596 1.596	3.0540 3.0424 3.0525 3.0677 3.0524	98.12 97.46 98.03 99.12 97.56

Average recovery rate=98.06%; RSD=0.67%.

Ginsenoside Rg ₁The average recovery test

Numbering	Test sample weighing (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	1.25042 1.24957 1.25038 1.24852 1.25662	7.3337 7.3287 7.3335 7.3226 7.3701	7.004 7.004 7.004 7.004 7.004	14.2565 14.1310 14.1225 14.0373 14.1226	98.84 97.12 96.93 95.87 96.41

Average recovery rate=97.03%; RSD=1.15%.

Ginsenoside Rb ₁The average recovery test

Numbering	Test sample weighing (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	1.25042 1.24957 1.25038 1.24852 1.25662	10.2935 10.2865 10.2931 10.2778 10.3445	9.728 9.728 9.728 9.728 9.728	19.8386 19.9201 19.8509 19.7247 19.7729	98.12 99.03 98.25 97.11 96.92

Average recovery rate=97.89%; RSD=0.89%.

12 3 batches of pilot scale sample sizes are measured

Three batch sample assay results

Lot number	Panax Notoginseng saponin R ₁(mg/ bottle)	Ginsenoside Rg ₁(mg/ bottle)	Ginsenoside Rb ₁(mg/ bottle)	Total saponins (mg/ bottle)
Lot number	Panax Notoginseng saponin R ₁(mg/ bottle)	Ginsenoside Rg ₁(mg/ bottle)	Ginsenoside Rb ₁(mg/ bottle)	Total saponins (mg/ bottle)	1 2 3	0.6552 0.6603 0.6621	3.012 3.008 3.275	4.358 4.271 4.304	8.025 7.939 8.241

The specific embodiment

Embodiment 1: adopt the liquid chromatography test to be characterized as main finger printing with the Radix Astragali flavone constituents

(1) preparation of need testing solution: get powder injection formulation 0.1g of the present invention, add the n-butyl alcohol dissolving, filter, filtrate evaporate to dryness, residue add methanol and are diluted to 5ml, as need testing solution;

(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 13 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;

(5) with said method as the means of testing that is characterized as main finger printing in the powder injection formulation of the present invention with the Radix Astragali flavone constituents, the preparation powder injection formulation of the present invention finger printing;

(6) with the finger printing and the contrast of above-mentioned standard finger-print of powder injection formulation of the present invention, meet the following requirements:

I. calculate the similarity of the finger printing and the standard finger-print of powder injection formulation of the present invention, should be 0.90～1.00;

II. in the powder injection formulation finger printing of the present invention, non-total peak area must not surpass 5% of total peak area;

III. the relative deviation of the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak must not surpass ± 10%.

Embodiment 2: adopt the liquid chromatography test to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents

(1) preparation of need testing solution: get powder injection formulation 0.1g of the present invention, add dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Astragali and the pseudo-ginseng, comprise the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁, a kind of in the astragaloside, use dissolve with methanol, be settled to suitable concn, as object of reference solution;

(5) with said method as the means of testing that is characterized as main finger printing in the powder injection formulation of the present invention with Radix Astragali saponin class, arasaponin constituents, the preparation testing sample finger printing;

Embodiment 3: adopt the liquid chromatography test to be characterized as main finger printing with the Radix Astragali flavone constituents

(1) preparation of need testing solution: it is an amount of to get powder injection formulation of the present invention, adds dissolve with methanol, filters, and filtrate evaporate to dryness, residue add dissolve with methanol or be diluted to suitable concn, as need testing solution;

(2) preparation of object of reference solution: get formononetin, use dissolve with methanol, be settled to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is methanol: 0.5% phosphoric acid solution, gradient elution, solvent ratios is from 0 minute to 30 minutes, the ratio of methanol is for to rise to 30% from 5%, from 30 minutes to 50 minutes, the ratio of acetonitrile is for to rise to 50% from 30%, from 50 minutes to 60 minutes, the ratio of acetonitrile is for rising to 70% from 50%, and from 60 minutes to 70 minutes, the ratio of methanol was for to rise to 85% from 70%, from 70 minutes to 90 minutes, the ratio of methanol is for rising to 100% from 85%, and flow velocity is 1.5ml/min, the detection wavelength is 250 ± 4nm, 60 ℃ of column temperatures;

(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 8～40;

(5) with said method as the means of testing that is characterized as main finger printing in the powder injection formulation of the present invention with the Radix Astragali flavone constituents, the preparation testing sample finger printing;

I. calculate the similarity of the finger printing and the standard finger-print of powder injection formulation of the present invention, should be 0.80～1.00;

II. in the powder injection formulation finger printing of the present invention, non-total peak area must not surpass 10% of total peak area;

III. the relative deviation of the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak must not surpass ± 20%.

Embodiment 4: adopt the liquid chromatography test to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents

(1) preparation of need testing solution: it is an amount of to get powder injection formulation of the present invention, adds ethanol dilution to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Astragali and the pseudo-ginseng, comprise the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁, a kind of in the astragaloside, use dissolve with ethanol, be settled to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is methanol: 2.5% glacial acetic acid, gradient elution, flow velocity are that 2.0ml/min, detection wavelength are 200 ± 2nm, 60 ℃ of column temperatures;

(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 10～40;

Embodiment 5: adopt the liquid chromatography test to be characterized as main finger printing with the Radix Astragali flavone constituents

(1) preparation of need testing solution: it is an amount of to get the little liquid drugs injection of the present invention, is dissolved in water, filter, and the filtrate evaporate to dryness, residue dissolve with ethanol or be diluted to suitable concn is as need testing solution;

(2) preparation of object of reference solution: get formononetin, use dissolve with ethanol, be settled to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile: 0.25% formic acid, gradient elution, flow velocity are that 0.5ml/min, detection wavelength are one or several in 252 ± 4nm scope, 20 ℃ of column temperatures;

(5) with said method as the means of testing that is characterized as main finger printing in the little liquid drugs injection of the present invention with the Radix Astragali flavone constituents, the preparation testing sample finger printing;

(6) with the finger printing and the contrast of above-mentioned standard finger-print of the little liquid drugs injection of the present invention, meet the following requirements:

I. calculate the similarity of the finger printing and the standard finger-print of the little liquid drugs injection of the present invention, should be 0.80～1.00;

II. in the little liquid drugs injection finger printing of the present invention, non-total peak area must not surpass 10% of total peak area;

Embodiment 6: adopt the liquid chromatography test to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents

(1) preparation of need testing solution: it is an amount of to get the little liquid drugs injection of the present invention, and thin up shakes up to suitable concn, filters, and gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Astragali and the pseudo-ginseng, comprise the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁, a kind of in the astragaloside, use water dissolution, be settled to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile: 0.5% phosphoric acid solution, gradient elution, flow velocity is 0.5ml/min, detect wavelength is that one or several or evaporative light scattering detector in 202 ± 4nm scope detects, and column temperature is in 20 ℃ of scopes;

(5) with said method as the means of testing that is characterized as main finger printing in the little liquid drugs injection of the present invention with Radix Astragali saponin class, arasaponin constituents, the preparation testing sample finger printing;

(6) with the finger printing of the little liquid drugs injection of the present invention and the contrast of above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:

Embodiment 7: the thin layer chromatography discrimination method of the Radix Astragali in the compound preparation

It is an amount of to get powder injection formulation of the present invention, adds ethanol extraction, filters the filtrate evaporate to dryness, residue adds the dissolving of 0.3% sodium hydroxide solution, filters, and filtrate is used ethyl acetate extraction with hydrochloric acid condition pH value to 5～6, filter, filtrate evaporate to dryness, residue add the ethyl acetate dissolving, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developing solvent with chloroform-methanol at 10: 1, launches, take out, dry, put in the ammonia steam and inspect under the smoked rearmounted ultra-violet lamp 365nm, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle.

Embodiment 8: the thin layer chromatography discrimination method of astragaloside in the compound preparation

It is an amount of to get powder injection formulation of the present invention, be added on the neutral alumina post after adding dissolve with methanol, use 40% methanol-eluted fractions, collect eluent, evaporate to dryness, add after residue is dissolved in water that water-saturated n-butanol extracts and and n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of 13: 7: 2 of chloroform-methanol-water is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts to reach under the ultra-violet lamp 365nm daylight under and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle.

Embodiment 9: the liquid chromatograph discrimination method of astragaloside in the compound preparation

It is an amount of to get the little hydro-acupuncture preparation of the present invention, the back that is dissolved in water water saturation n-butanol extraction and extracting solution also, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol or 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 10: Radix Notoginseng, ginsenoside Rg in the compound preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more thin layer chromatography discrimination method:

It is an amount of to get powder injection formulation of the present invention, uses n-butanol extraction, filters, and filtrate is as need testing solution; Other gets Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more, preparation control medicinal material solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adds the chloroform reflux, extract,, discards chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: 10 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless.

Embodiment 11: ginsenoside Rg in the compound preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more liquid chromatograph differentiate

It is an amount of to get powder injection formulation of the present invention, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Re, Panax Notoginseng saponin R ₁In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

Embodiment 12: the thin layer chromatography discrimination method of the Radix Astragali in the compound preparation

It is an amount of to get powder injection formulation of the present invention, adds ethyl acetate extraction, filters the filtrate evaporate to dryness, residue adds the dissolving of 2.5% sodium hydroxide solution, filters, and filtrate is used ethyl acetate extraction with hydrochloric acid condition pH value to 3, filter, filtrate evaporate to dryness, residue add the ethyl acetate dissolving, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel H lamellae, be developing solvent with dichloromethane-ethanol at 15: 7, launches, take out, dry, put in the ammonia steam and inspect under the smoked rearmounted daylight, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;

Embodiment 13: the thin layer chromatography discrimination method of astragaloside in the compound preparation

It is an amount of to get the little liquid drugs injection of the present invention, be added on the neutral alumina post after adding dissolve with ethanol, use 60% methanol-eluted fractions, collect eluent, evaporate to dryness, add water-saturated n-butanol after residue is dissolved in water and extract, merge n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 8 μ l of above-mentioned solution, put respectively on same silica gel polyamide film, with lower floor's solution of 10: 8: 5 of dichloromethane-ethanol-water is developing solvent, launch, take out, dry, spray is with 8% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts under the daylight and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;

The liquid chromatograph discrimination method of astragaloside in embodiment 14, the compound preparation

It is an amount of to get capsule of the present invention, and back ethyl acetate extraction, merge extractive liquid, are dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, ethyl acetate liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Alcoholic solution with the astragaloside reference substance is contrast, adopts liquid chromatography, and chromatographic column is a filler with eight alkyl silane bonded silica gels, and methanol-5% glacial acetic acid aqueous solution was a mobile phase in 20%: 80%, detects wavelength 360nm and detects 60 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 15: Radix Notoginseng, ginsenoside Rg in the compound preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more thin layer chromatography discrimination method

It is an amount of to get the little liquid drugs injection of the present invention, uses methanol extraction, filters, and filtrate is as need testing solution; Other gets Radix Notoginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more, the preparation contrast solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, and the reflux, extract, that adds diethyl ether discards ether solution, the residue ethyl acetate extraction, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances, add ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 12 μ l of above-mentioned solution, put in silica gel G F respectively ₂₅₄On the lamellae, with n-butyl alcohol-Ethyl formate-upper strata liquid of 5: 1: 8 of water is developing solvent, launches, and takes out, dry, spray is with 50% sulphate reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color, negative noiseless.

Embodiment 16: ginsenoside Rg in the compound preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more liquid chromatograph differentiate

It is an amount of to get the little liquid drugs injection of the present invention, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Re, Panax Notoginseng saponin R ₁In the alcoholic solution of one or more reference substances be contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, methanol-2.5% formic acid solution was a mobile phase in 25%: 75%, and gradient elution, flow velocity are 2.0ml/min, the evaporation photodetector detects, and column temperature is at 60 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

Embodiment 17: the assay of astragaloside in the compound preparation

It is an amount of to get glucose infusion liquid preparation of the present invention, the back that is dissolved in water water saturation n-butanol extraction and extracting solution also, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, glucose infusion liquid preparation of the present invention is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.04mg.

Embodiment 18: ginsenoside Rg in the compound preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more assay

It is an amount of to get powder injection formulation of the present invention, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with external standard method or standard curve method, powder injection formulation of the present invention is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(4) the per unit amount contains the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The limit of summation must not be less than 12mg.

Embodiment 19: the assay of total saponins in the compound preparation

It is an amount of to get powder injection formulation of the present invention, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision measures 0.6, puts in the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with the ginsenoside Rg ₁Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, compound preparation to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg ₁Meter must not be less than 20mg.

Embodiment 20: the assay of astragaloside in the compound preparation

It is an amount of to get powder injection formulation of the present invention, and back ethyl acetate extraction, merge extractive liquid, are dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Alcoholic solution with the astragaloside reference substance is contrast, adopts liquid chromatography, and chromatographic column is a filler with the dialkyl silane bonded silica gel, and methanol-5.0% glacial acetic acid aqueous solution was a mobile phase in 40%: 60%, detects wavelength 203nm and detects 20 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, powder injection formulation of the present invention is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains must not be less than 0.02mg;

Embodiment 21: ginsenoside Rg in the compound preparation ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more assay:

It is an amount of to get the little liquid drugs injection of the present invention, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In the alcoholic solution of one or more reference substances be contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, methanol-3.0% phosphate aqueous solution was a mobile phase in 15%: 85%, and gradient elution, flow velocity are 2.0ml/min, the evaporation photodetector detects 60 ℃ of column temperatures; Calculate with external standard method or standard curve method, the little liquid drugs injection of the present invention is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(4) the per unit amount contains the ginsenoside Rg ₁Limit must not be less than 2.5mg;

(5) the per unit amount contains ginsenoside Rb ₁Limit must not be less than 3.5mg;

(6) the per unit amount contains Panax Notoginseng saponin R ₁Limit must not be less than 0.25mg;

The per unit amount contains the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The limit of summation must not be less than 6mg.

Embodiment 22: the assay of total saponins in the compound preparation

It is an amount of to get dispersible tablet of the present invention, puts in the measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured in right amount, puts in the measuring bottle, water bath method takes out immediately, and precision adds 15% vanillin-glacial acetic acid solution 0.1ml, perchloric acid 1ml shakes up, and heating is 30 minutes in 40 ℃ of water-baths, take out, with the flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with astragaloside or ginsenoside Rg ₁Or ginsenoside Rb ₁Or Panax Notoginseng saponin R ₁Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures trap, calculate with one point external standard method or standard curve method, dispersible tablet of the present invention is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with astragaloside or ginsenoside Rg ₁Or ginsenoside Rb ₁Or Panax Notoginseng saponin R ₁Meter must not be less than 10mg.

Embodiment 23: formononetin in the compound preparation, hair stamen formononetin assay

It is an amount of to get powder injection formulation of the present invention, adds methanol extraction, and extracting solution is as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, powder injection formulation of the present invention is unit quantity to be equivalent to every day with output, and content limit should be:

Embodiment 24: formononetin in the compound preparation, hair stamen formononetin assay

It is an amount of to get drop pill of the present invention, adds ethanol extraction, and extracting solution is as need testing solution; Alcoholic solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and chromatographic column is a filler with the dialkyl silane bonded silica gel, mobile phase A is an acetonitrile, and Mobile phase B is that 0.5% phosphate aqueous solution is a mobile phase, gradient elution, the detection wavelength is 255nm, 50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, drop pill of the present invention is unit quantity to be equivalent to every day with output, and content limit should be:

Claims

1, a kind of method of quality control of the compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng, it is characterized in that: this method comprises following all or part of content:

2, according to the method for quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng of claim 1, it is characterized in that: this method adopts liquid chromatography test to be characterized as main finger printing and to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents:

(1) preparation of need testing solution: it is an amount of to get compound preparation to be measured, adds the dissolving of water or methanol or ethanol or n-butyl alcohol, filters, and filtrate evaporate to dryness, residue add methanol or dissolve with ethanol or be diluted to suitable concn, as need testing solution;

3, according to the method for quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng of claim 2, it is characterized in that: this method adopts liquid chromatography test to be characterized as main finger printing and to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents:

(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the compound preparation to be measured with the Radix Astragali flavone constituents, the preparation testing sample finger printing;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile: water, gradient elution,, solvent ratios is from 0 minute to 12 minutes, the ratio of acetonitrile is 15%, from 14 minutes to 45 minutes, the ratio of acetonitrile is for rising to 30% from 15%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was for to rise to 80% from 30%, flow velocity is 1ml/min, detection wavelength 203 ± 2nm, 30 ℃ of column temperatures;

4, according to the method for quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng of claim 1, it is characterized in that: the discrimination method of described compound Chinese medicinal preparation comprises one or more in following:

5, according to the method for quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng of claim 4, it is characterized in that: the discrimination method of described compound Chinese medicinal preparation comprises one or more in following:

It is an amount of to get compound preparation to be measured, be added on the neutral alumina post after adding dissolve with methanol, use 40% methanol-eluted fractions, collect eluent, evaporate to dryness, add after residue is dissolved in water that water-saturated n-butanol extracts and and n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of 13: 7: 2 of chloroform-methanol-water is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts to reach under the ultra-violet lamp 365nm daylight under and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;

It is an amount of to get compound preparation to be measured, the back that is dissolved in water water saturation n-butanol extraction and extracting solution also, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol or 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 32%: 68%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

6, according to the method for quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng of claim 1, it is characterized in that: the method for testing of described compound Chinese medicinal preparation content should comprise one or more in following:

The assay of astragaloside in a, the compound Chinese medicinal preparation:

The assay of total saponins in c, the compound Chinese medicinal preparation:

7, according to the method for quality control of the described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng of claim 6, it is characterized in that: the method for testing of described compound Chinese medicinal preparation content is following one or more methods:

The assay of astragaloside in a, the compound Chinese medicinal preparation:

It is an amount of to get compound preparation to be measured, the back that is dissolved in water water saturation n-butanol extraction and extracting solution also, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 32%: 68%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.04mg;

The assay of total saponins in c, the compound Chinese medicinal preparation:

8, according to the method for quality control of claim 6 or the 7 described compound Chinese medicinal preparation made from the Radix Astragali, Radix Notoginseng, it is characterized in that: the assay result of described ejection preparation, calculate astragaloside, formononetin, calycosin, total saponins, Panax Notoginseng saponin R according to percentage by weight ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁In the total content of all or part of material account for deduction adjuvant and outer more than 25% of total solid of moisture in the preparation.