CN1951426A

CN1951426A - Quality control method of a compound Chinese medicinal preparation

Info

Publication number: CN1951426A
Application number: CN 200510109394
Authority: CN
Inventors: 于文风
Original assignee: Qiyuanyide Medicines Institute Beijing
Current assignee: Qiyuanyide Medicines Institute Beijing
Priority date: 2005-10-19
Filing date: 2005-10-19
Publication date: 2007-04-25

Abstract

The invention relates to a method of quality control for Chinese compound medicinal compositions prepared from herba erigerontis, pseudo-ginseng, astragalus root or the extract and significant portions of them, which includes fingerprint pattern testing process and/or discrimination testing process and/or content determination process for herba erigerontis, astragalus root, pseudo-ginseng in the preparation. The invention provides a reliable, stable and novel method for quality control of the preparation, which realizes more stricter and reasonable process control, more stable quality, more reliable detection method for mass production, and digitalized illustration for guaranteeing safety and effectiveness of patient's drug administration is provided.

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 scutellarin, astragaloside, Panax Notoginseng saponin R ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁In certain composition for detecting index, but its quality of reactor product comprehensively at all is 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):

Be made for 1～100 part by 10～500 parts of Herba Erigerontiss, 1～100 part of the Radix Astragali and Radix Notoginseng; Or add suitable adjuvant and be made through extracting the extract that obtains after refining by corresponding weight portion medical material.

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 compound Chinese medicinal preparation is characterized in that: this method comprises following all or part of content:

(1) finger printing test comprises with Herba Erigerontis, 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) Herba Erigerontis control medicinal material, Radix Astragali control medicinal material, Radix Notoginseng control medicinal material, scutellarin, 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) scutellarin, astragaloside, formononetin, calycosin, Panax Notoginseng saponin R ₁, the ginsenoside Rg ₁, ginsenoside Rb ₁, the content test method of all or part of composition in the total saponins, total flavones.

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

A, the test of employing liquid chromatography are characterized as main finger printing with Herba Erigerontis, 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 water or methanol or dissolve with ethanol or dilution, 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 Herba Erigerontis and the Milkvetch Root, comprise a kind of in scutellarin, formononetin, the calycosin, 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 0.5%～100% acetonitrile solution or methanol-0.005mol/L～5mol/L sodium dihydrogen phosphate or 0.005mol/L～5mol/L potassium dihydrogen phosphate 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 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 Herba Erigerontis, 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 Herba Erigerontis, Radix Astragali flavone constituents, the preparation testing sample finger printing;

(6) with the finger printing of compound Chinese medicinal 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 Chinese medicinal preparation to be measured, should be 0.80～1.00;

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

III. the total chromatographic peak of other except that the object of reference chromatographic peak is compared with standard finger-print with the relative retention time of object of reference chromatographic peak, 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 Chinese medicinal 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 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 Chinese medicinal preparation to be measured with Radix Astragali saponin class, arasaponin constituents, the preparation testing sample finger printing;

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%.

A, employing liquid chromatography for measuring are characterized as main finger printing with Herba Erigerontis, Radix Astragali flavone constituents:

(1) preparation of need testing solution: it is an amount of that precision takes by weighing compound Chinese medicinal preparation to be measured, adds water and make the solution that every 1ml contains 1mg, shakes up, promptly;

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing scutellarin, adds methanol and make the solution that every 1ml contains 0.1mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is 80% acetonitrile, and Mobile phase B is 0.1% phosphoric acid solution, gradient elution, and solvent ratios was from 0 minute to 80 minutes, and the ratio of mobile phase A rises to 70% by 15%, and the ratio of Mobile phase B reduces to 30% by 85%; Flow velocity is 1.0ml/min, and the detection wavelength is 230 ± 2nm, and column temperature is 40 ℃;

(4) formulation of standard finger-print: the means of testing that is characterized as main standard finger-print with said method as formulation with Herba Erigerontis, Radix Astragali flavone constituents; Accurate respectively object of reference solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, according to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, retention time with definite object of reference chromatographic peak is a benchmark, calculate 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 Chinese medicinal preparation to be measured, should be 0.90～1.00;

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

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

(1) preparation of need testing solution: it is an amount of that precision takes by weighing compound Chinese medicinal preparation to be measured, adds methanol and make the solution that every 1ml contains 50mg, shakes up, promptly;

(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg ₁In right amount, add methanol and make the solution that every 1ml contains 0.3mg, 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 14 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 3～20;

(5) with the described method in (1)～(3) as in the compound Chinese medicinal preparation to be measured based on the means of testing of the finger printing of Radix Notoginseng composition characteristics, the finger printing of preparation testing sample;

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

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

One or more thin layer chromatography discrimination method in Herba Erigerontis medical material, the scutellarin in a, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal preparation to be measured, adds ethyl acetate or ethanol or methanol or n-butanol extraction, and filtration or centrifugal is got filtrate or supernatant as need testing solution; Other gets one or both preparation contrast solutions in Herba Erigerontis control medicinal material, the scutellarin reference substance; The preparation of Herba Erigerontis control medicinal material solution: get the Herba Erigerontis control medicinal material, add ethyl acetate or ethanol or methanol or n-butanol extraction, filter, filtrate volatilizes, and residue is with methanol or dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the scutellarin reference substance, add methanol or ethanol and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw above-mentioned need testing solution and Herba Erigerontis control medicinal material solution, each 1～30 μ l of one or both of scutellarin reference substance solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or polyamide film, with ethyl acetate or Ethyl formate-formic acid or acetic acid-water or methanol 0.2～60: 0.2～10: 0.3～20 or benzene or toluene-ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1～30: 0.5～15: 0.05～5: 0.01～3 is developing solvent, launch, take out, dry, spray is with 0.3%～10% aluminum chloride ethanol or 0.3～10% aluminum chloride methanol solution or 0.3～10% ferric chloride alcoholic solution, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect or 1～15 minute rearmounted daylight of 105 ℃ of bakings is inspected down or under ultra-violet lamp 365nm or the 254nm, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the same color speckle;

The liquid chromatograph discrimination method of scutellarin in b, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal preparation to be measured, adds methanol or ethanol or mobile phase or water dissolution or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Methanol or alcoholic solution with the scutellarin 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, with 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 5%～95%: 95%～5% or methanol or acetonitrile-oxolane-0.01%～5% phosphate aqueous solution 2%～30%: 2%～30%: 96%～40% or acetonitrile-0.005moL/L～0.3moL/L sodium dihydrogen phosphate (1～99% phosphoric acid is transferred pH=2.0～5.0) gradient elution system is mobile phase, and the detection wavelength is 200～410nm; 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 c, 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 Chinese medicinal 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 in Radix Notoginseng control medicinal material, ginsenoside Rg1, ginsenoside Rb1, the arasaponin R1 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 on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 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 control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color;

Ginsenoside Rg in d, 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 Chinese medicinal 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 5%～40%: 95%～60% 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;

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

It is an amount of to get compound Chinese medicinal 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 with hydrochloric acid condition pH value to 3～6, 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 on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or 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 f, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal 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 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 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or 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 g, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal preparation to be measured, and it is back with n-butyl alcohol or ethyl acetate extraction, merge extractive liquid, to be 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 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 a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 15%～85%: 85%～15% 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 are mobile phase, 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;

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

It is an amount of to get compound Chinese medicinal 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 on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 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 i, the compound Chinese medicinal preparation, hair stamen formononetin:

It is an amount of to get compound Chinese medicinal 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.

It is an amount of to get compound Chinese medicinal preparation to be measured, adds methanol extraction, centrifugal, gets supernatant as need testing solution; Other gets one or more preparation contrast solutions in Herba Erigerontis control medicinal material, the scutellarin; The preparation of Herba Erigerontis control medicinal material solution: get the Herba Erigerontis control medicinal material, add ethyl acetate extraction, filter, filtrate volatilizes, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the scutellarin reference substance, add methanol and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, put on same polyamide membrane with strip tape respectively, be developing solvent with methanol-ethyl acetate-formic acid at 7: 2: 1, launches, take out, dry, spray is with 2% ferric chloride alcoholic solution, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the streak of same color;

It is an amount of to get compound Chinese medicinal preparation to be measured, adds dissolve with methanol or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution is a mobile phase at 50%: 50%, and the detection wavelength is 335nm; 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 Chinese medicinal preparation to be measured, uses n-butanol 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, 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 one or more reference substances in ginsenoside Rg1, ginsenoside Rb1, the arasaponin R1, 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, 105 ℃ are dried by the fire to 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;

It is an amount of to get compound Chinese medicinal 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 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;

It is an amount of to get compound Chinese medicinal 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 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;

It is an amount of to get compound Chinese medicinal 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 Chinese medicinal preparation to be measured, and back water saturation n-butanol 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 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 Chinese medicinal 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 Chinese medicinal 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 Herba Erigerontis, Radix Notoginseng, the Radix Astragali 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 scutellarin in a, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal preparation to be measured, adds methanol or ethanol or mobile phase or water dissolution or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Methanol or alcoholic solution with the scutellarin 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, with 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 5%～95%: 95%～5% or methanol or acetonitrile-oxolane-0.01%～5% phosphate aqueous solution 2%～30%: 2%～30%: 96%～40% or acetonitrile-0.005moL/L～0.3moL/L sodium dihydrogen phosphate (1～99% phosphoric acid is transferred pH=2.0～5.0) gradient elution system is mobile phase, and the detection wavelength is 200～410nm; 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 scutellarin must not be less than 2mg;

Content of total flavone is measured in b, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal preparation to be measured, with methanol or water dissolution and be diluted to suitable concn, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system; With the retinue solvent is blank, adopts the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measures trap at the wavelength place of 335 ± 10nm, calculates with one point external standard method or standard curve method; Or it is an amount of to get compound Chinese medicinal preparation to be measured, is dissolved in water and is diluted to suitable concn, shakes up, precision is measured in right amount, puts in the measuring bottle, adds 50% methanol or water 1～25ml, add 5% sodium nitrite solution, 0.3～1ml, shake up, placed 6 minutes, add 10% aluminum nitrate solution, 0.3～1ml, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4～10ml, add 50% methanol or water again to scale, shake up, as need testing solution; With rutin or scutellarin product in contrast, get reference substance solution with legal system; With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap at 500 ± 10nm place, calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains the limit of total flavones in rutin or scutellarin, must not be less than 5mg;

Ginsenoside Rg in c, 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 Chinese medicinal 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 external standard method or standard curve method, compound Chinese medicinal 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:

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

The per unit amount contains ginsenoside Rb ₁Limit must not be less than 3.5mg;

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 d, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal preparation to be measured, put in the measuring bottle, adding distil water makes dissolving in right amount and decides and shakes up to scale, and precision is measured in right amount, put 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, shake up, heated 3～50 minutes in 30～80 ℃ of water-baths, take out, immediately with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale, shakes up, as need testing solution, with astragaloside or ginsenoside Rg1 or ginsenoside Rb1 or arasaponin R1 is reference substance, gets 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 Chinese medicinal 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 astragaloside in e, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal preparation to be measured, and it is back with n-butyl alcohol or ethyl acetate extraction, merge extractive liquid, to be 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; 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, 15%～85%: 85%～15% 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 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 astragaloside must not be less than 0.02mg;

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

It is an amount of to get compound Chinese medicinal 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 Chinese medicinal 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 Herba Erigerontis, Radix Notoginseng, the Radix Astragali, 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 scutellarin in a, the compound Chinese medicinal preparation:

It is an amount of to get compound Chinese medicinal preparation to be measured, adds dissolve with methanol or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution is a mobile phase at 50%: 50%, and the detection wavelength is 335nm; A bit calculate with external standard, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg;

It is an amount of to get compound Chinese medicinal preparation to be measured, puts in the measuring bottle, adds methanol and makes dissolving and fixed to scale in right amount, shakes up, and precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at the wavelength place of 335nm, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the limit of total flavones in scutellarin, must not be less than 10mg;

It is an amount of to get compound Chinese medicinal 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 external standard method or standard curve method, compound Chinese medicinal 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 d, 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 Chinese medicinal 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;

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

It is an amount of to get compound Chinese medicinal preparation to be measured, and back water saturation n-butanol 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; 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 Chinese medicinal 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 30 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, compound Chinese medicinal 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 Herba Erigerontis, Radix Notoginseng, the Radix Astragali, it is characterized in that: the assay result of described ejection preparation, calculate scutellarin, astragaloside, formononetin, calycosin, ginsenoside Rg according to percentage by weight ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁, all or part of material in the total saponins, total flavones total content 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 Herba Erigerontis, Radix Notoginseng, the Radix Astragali 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 Herba Erigerontis, 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 in the compound Chinese medicinal preparation being characterized as main finger printing and being characterized as main finger printing characteristic peak changing with Radix Astragali saponin class, arasaponin constituents with Herba Erigerontis, Radix Astragali flavone constituents, 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 Herba Erigerontis, Radix Notoginseng, the Radix Astragali, and method precision, stability are all higher.

Experimental example 1 is characterized as the preparation of main finger printing with Herba Erigerontis, Radix Astragali flavone constituents

A, experimental apparatus, reagent and sample:

Reference substance: scutellarin: 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, can reach 70000 (calculating with the object of reference scutellarin).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-water (20: 80) in the research process respectively, (2) acetonitrile-water (10: 90), (3) acetonitrile-water (gradient elution) (4) mobile phase A is 80% acetonitrile, Mobile phase B is 0.1% phosphoric acid solution, gradient elution, solvent ratios was from 0 minute to 80 minutes, and the ratio of mobile phase A rises to 70% by 15%, and the ratio of Mobile phase B rises to 30% 4 kind of flow phase system by 85%.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:

Be 80% acetonitrile in mobile phase A in the research, Mobile phase B is 0.1% phosphoric acid solution, under the gradient elution mobile phase condition, investigated the chromatographic peak situation under different-waveband typical wavelengths 230,254,270,300 respectively, the result shows, 230nm can take into account kurtosis, separating degree and the baseline of each composition chromatographic peak when detecting, so select 230nm 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: 50000.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 1mg, promptly.

6. the preparation of object of reference solution:

It is an amount of that precision takes by weighing scutellarin, adds 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 (with the object of reference ginsenoside Rg ₁Calculate).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 14 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:

One or more thin layer chromatography discrimination method in Herba Erigerontis medical material, the scutellarin in experimental example 3 compound Chinese medicinal preparation:

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

Condition question

Normal hexane-benzene-Ethyl formate-formic acid (5-5-10-4) silica gel H lamellae reference substance is expanded to the forward position

Ethyl acetate-benzene-acetic acid (5-4-3) silica gel H lamellae reference substance does not separate, and feminine gender has interference

Ethyl acetate-benzene-formic acid (5-4-3) silica gel g thin-layer plate reference substance does not separate, and feminine gender has interference

Chloroform-ethyl acetate-methanol (7-2-4) silica gel G F ₂₅₄The lamellae reference substance does not separate, and feminine gender has interference

Toluene-ethyl acetate (8-1) silica gel H lamellae reference substance does not separate, and feminine gender has interference

Methanol-ethyl acetate-formic acid (10-5-0.5) silica gel g thin-layer plate reference substance is expanded to the forward position

It is clear that methanol-ethyl acetate-formic acid (7-2-1) polyamide membrane is separated, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the polyamide membrane to be immobile phase, be developing solvent with methanol-ethyl acetate-formic acid (7-2-1), with this understanding, the Rf value of scutellarin is moderate, and it is clear to separate with other speckle, negative noiseless.

The liquid chromatograph discrimination method of scutellarin in experimental example 4 compound Chinese medicinal preparation:

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

Condition question

Methanol-0.05mol/L sodium hydrogen phosphate (80: 20) eight alkyl silane bonded silica gel appearance times are too fast

Acetonitrile-0.05mol/L sodium hydrogen phosphate (40: 60) octadecylsilane chemically bonded silica feminine gender has interference

Methanol-oxolane-0.05mol/L sodium hydrogen phosphate (20: 10: 70) octadecyl feminine gender has interference

Silane group silica gel

Acetonitrile-oxolane-0.05mol/L sodium hydrogen phosphate (20: 10: 70) octadecyl peak shape is slightly asymmetric

Silane group silica gel

Methanol-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane feminine gender has interference

Bonded silica gel

Acetonitrile-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane peak shape is slightly asymmetric

Bonded silica gel

Retention time is moderate, the capable point in peak

Methanol-0.1% phosphate aqueous solution (50: 50) octadecylsilane chemically bonded silica is sharp, and symmetry is negative noiseless

Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, methanol-0.1% phosphate aqueous solution (50: 50) is a mobile phase, and with this understanding, the scutellarin retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless.

Experimental example 5 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 question

The lower floor that chloroform-Ethyl formate-water (20: 60: 10) is placed below 10 ℃ is molten

Reference substance is expanded to the forward position

The liquid silica gel g thin-layer plate

N-butyl alcohol-Ethyl formate-methanol (10-1-5) silica gel H lamellae reference substance is expanded to the forward position

Chloroform-Ethyl formate-formic acid-water (15: 40: 22: 10) place below 10 ℃

Reference substance does not separate, and feminine gender has interference

Lower floor's solution silica gel g thin-layer plate

Chloroform-ethyl acetate-formic acid-water (10: 40: 15: 5) place below 10 ℃

Reference substance does not separate, and feminine gender has interference

Lower floor's solution silica gel g thin-layer plate

N-butyl alcohol-Ethyl formate-methanol (5-1-5) silica gel g thin-layer plate reference substance does not separate, and feminine gender has interference

N-butyl alcohol-Ethyl formate-ethanol (2-1.5-0.5) silica gel H lamellae reference substance does not separate, and feminine gender has interference

Determine condition: chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) 10

It is clear to separate, and the moderate feminine gender of Rf value is noiseless

Lower floor's solution silica gel g thin-layer plate of placing below ℃

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 6 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 question

Methanol-0.05mol/L sodium dihydrogen phosphate (70: 30) octadecylsilane chemically bonded silica appearance time is too fast

Acetonitrile-0.05mol/L sodium dihydrogen phosphate (55: 45) octadecylsilane chemically bonded silica appearance time is too fast

Methanol-0.05mol/L sodium hydrogen phosphate (80: 20) eight alkyl silane bonded silica gel feminine genders have interference

Methanol-0.05mol/L sodium hydrogen phosphate (80: 20) octadecylsilane chemically bonded silica feminine gender has interference

Acetonitrile-0.02mol/L potassium dihydrogen phosphate (90: 10) eight alkyl silane bonded silica gel feminine genders have interference

Methanol-0.02mol/L potassium dihydrogen phosphate (85: 15) octadecylsilane chemically bonded silica feminine gender has interference

Acetonitrile-water is a mobile phase, gradient elution, and 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 was moderate by retention time on 20%, the capable point in peak

Rise to 40%, from 21 minutes to 25 minutes, the ratio of acetonitrile was that 20% octadecyl silicon is sharp, and symmetry is negative noiseless

The alkane bonded silica gel

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.

The thin layer chromatography discrimination method of the Radix Astragali in experimental example 7 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 question

N-butyl alcohol-glacial acetic acid-water (6: 4: 3) silica gel H lamellae feminine gender has interference

Acetone-glacial acetic acid-water (8: 2: 1) silica gel g thin-layer plate feature speckle is unintelligible

Methylene chloride-methanol (7-3) silica GF254 lamellae feminine gender has interference

Chloroform-acetone-formic acid (20: 2: 1) silica gel g thin-layer plate feminine gender has interference

Chloroform-acetone-methanol (20: 3: 2) silica gel H lamellae feminine gender has interference

Chloroform-acetone-methanol (20: 2: 10) silica gel g thin-layer plate reference substance is expanded to the forward position

It is clear that chloroform-methanol (10-1) silica gel g thin-layer plate separates, 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 8 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 question

Chloroform-acetone-water (10-8-0.5) silica gel g thin-layer plate feminine gender has interference

Dichloromethane-acetone-water (10-8-0.5) silica gel g thin-layer plate feminine gender has interference

The exhibition of dichloromethane-acetone (2-9) silica gel H lamellae reference substance is to the forward position

Chloroform-acetone (10-5) silica GF254 lamellae feminine gender has interference

Chloroform-ethanol (13-7) silica gel H lamellae feminine gender has interference

Chloroform-methanol (13-7) silica gel g thin-layer plate feminine gender has interference

Chloroform-methanol-water (13-7-2) lower floor solution separating is 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 9 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 question

Methanol-0.02mol/L sodium hydrogen phosphate (70: 30) eight alkyl silane bonded silica gel appearance times are too fast

Acetonitrile-0.02mol/L sodium hydrogen phosphate (50: 50) octadecylsilane chemically bonded silica appearance time is too fast

Methanol-0.05mol/L sodium hydrogen phosphate (40: 60) octadecylsilane chemically bonded silica feminine gender has interference

Acetonitrile-0.05mol/L sodium hydrogen phosphate (30: 70) octadecylsilane chemically bonded silica peak shape is slightly asymmetric

Methanol-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane

Feminine gender has interference

Bonded silica gel

Acetonitrile-methanol-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane bonding

Peak shape is slightly asymmetric

Silica gel

Retention time is moderate, the peak

Acetonitrile-water (32: 68) octadecylsilane chemically bonded silica is capable sharp-pointed, symmetry, and negative do not have

Disturb

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.

The thin layer chromatography discrimination method of formononetin, hair stamen formononetin in experimental example 10 compound Chinese medicinal preparation

Feature for outstanding Radix Astragali flavone constituents, selected formononetin, hair stamen formononetin as its feature speckle, but because have in the compound Chinese medicinal preparation that more and formononetin, hair stamen formononetin structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition launches formononetin, hair stamen formononetin:

Condition question

Benzene-acetone-methanol (5-3-1) silica gel g thin-layer plate feminine gender has interference

Toluene-ethyl acetate-water (10-8-0.5) silica gel g thin-layer plate feminine gender has interference

Normal hexane-methanol (6-0.5) silica gel H lamellae feminine gender has interference

The exhibition of cyclohexane extraction-acetone (5-5) silica GF254 lamellae reference substance is to the forward position

Benzene-Ethyl formate-methanol (6-2-1) silica gel H lamellae feminine gender has interference

Toluene-normal hexane-methanol (8-4-4) silica gel g thin-layer plate feminine gender has interference

Chloroform-methanol-ethyl acetate-water (2.5-3-4-0.5) silica gel

It is clear that the G lamellae separates, 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-ethyl acetate-water (2.5-3-4-0.5), with this understanding, the Rf value of formononetin, hair stamen formononetin feature speckle is moderate, and it is clear to separate with other speckle, negative noiseless.

The liquid chromatograph discrimination method of formononetin, hair stamen formononetin in experimental example 11 compound Chinese medicinal preparation

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

Condition question

Methanol-water (70: 30) eight alkyl silane bonded silica gel appearance times are too fast

Acetonitrile-water (50: 50) octadecylsilane chemically bonded silica appearance time is too fast

Methanol-1% glacial acetic acid (40: 60) octadecylsilane chemically bonded silica feminine gender has interference

Acetonitrile-0.5% formic acid (30: 70) octadecylsilane chemically bonded silica appearance time is slow excessively

Methanol-0.1% phosphoric acid (20: 10: 70) octadecylsilane chemically bonded silica feminine gender has interference

Acetonitrile-0.02mol/L sodium dihydrogen phosphate (30: 70) octadecylsilane chemically bonded silica feminine gender has interference

Methanol-water is a mobile phase, gradient elution, and solvent ratios is from 0 minute to 10 minutes,

The ratio of water rises to 50% by 40%, and from 10 minutes to 30 minutes, the ratio of water was moderate by 50% retention time, the capable point in peak

Rise to 60%, from 30 minutes to 50 minutes, the ratio of water was that 60% octadecylsilane key is sharp, and symmetry is negative noiseless

Close silica gel

Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, methanol-water is a mobile phase, gradient elution, solvent ratios are that the ratio of water rose to 50% by 40% from 0 minute to 10 minutes, from 10 minutes to 30 minutes, the ratio of water rose to 60% by 50%, from 30 minutes to 50 minutes, the ratio of water is 60% for mobile phase, with this understanding, formononetin, hair stamen formononetin retention time are moderate, and the peak is capable sharp-pointed, symmetry, negative noiseless.

Scutellarin assay in experimental example 12 compound Chinese medicinal preparation

1 instrument and reagent

1.1 key instrument:

High performance liquid chromatograph LC-2010AHT SHIMADZU

Ultraviolet/general all purpose instrument the company limited of analysing in visible spectrophotometer TU-1810SPC Beijing

Electronic analytical balance BP211D SARTORIUS

Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.

1.2 reagent:

Scutellarin Nat'l Pharmaceutical ﹠ Biological Products Control Institute

Methanol analytical pure Beijing Chemical Plant

Acetonitrile chromatographically pure Di Ma company

The pure Beijing of phosphate analysis chemical reagents corporation

2 scutellarins provide scutellarin by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and measuring purity through high performance liquid chromatography (normalization method) is 96.40%, meet assay with reference substance requirement (in mensuration, converting according to 96.40% purity).

It is an amount of that the scutellarin reference substance is got in 3 selections that detect wavelength, and accurate the title decides, and adds methanol and makes the solution that every 1ml contains 0.0463mg, in the interscan of 190～400nm wave-length coverage.The result shows that scutellarin has absorption maximum at 284nm and 335nm place, according to " the Sanitation Ministry medicine standard Chinese traditional patent formulation preparation " relevant kind and in conjunction with bibliographical information, selects the detection wavelength of 335nm as the scutellarin assay.

4 chromatographic conditions

Chromatograph: SHIMADZU LC-2010AHT;

Chromatographic column: Diamonsil ODS 250mm * 4.6mm 5 μ m;

Mobile phase: methanol-0.1% phosphate aqueous solution (50: 50);

Flow velocity: 1.0ml/min;

Column temperature: 30 ℃;

Sample size: 10 μ l;

Detect wavelength: 335nm.

It is an amount of that the preparation precision of reference substance solution takes by weighing the scutellarin reference substance, adds methanol and make the solution that every 1ml contains 0.05mg, promptly.

This product under the content uniformity item is got in the preparation of need testing solution, gets content, mixing, therefrom get about 0.25g, the accurate title, decide, and puts in the 25ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m).

Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Obtain scutellarin reference substance, test sample chromatogram according to above-mentioned chromatographic condition, its number of theoretical plate n is all greater than 2000, and the scutellarin chromatographic peak separates clear complete with close peak, and separating degree is all greater than 1.5, and solvent is noiseless.

This product under the content uniformity item is got in the selection of 5 extraction times, gets content, mixing, therefrom get about 0.25g (totally 3 parts), the accurate title, decide, and splits in the 25ml measuring bottle, it is an amount of to add methanol, respectively supersound process (power 250W, frequency 33KHz) 5,10,20min, take out, put, add methanol to scale to room temperature, shake up, filter, the accurate subsequent filtrate 10 μ l that draw with microporous filter membrane (0.45 μ m), inject chromatograph of liquid, measure, promptly.

Extraction time is investigated

Extraction time (min)	Content (mg/ bottle)
Extraction time (min)	Content (mg/ bottle)	5 10 20	2.541 2.537 2.546

The result shows that supersound process 5min can extract fully.

6 linear relationships investigation precision is measured scutellarin reference substance solution (C=0.978mg/ml) 0.0,0.2,0.4,0.6,0.8,1.0ml, split in the 10ml measuring bottle, be diluted to scale with methanol, shake up, the therefrom accurate respectively 10 μ l that draw, inject chromatograph of liquid, measure according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000).With the peak area is vertical coordinate, and the amount of scutellarin (μ g) is abscissa mapping, drawing standard curve.

Scutellarin standard curve determination result

Numbering	Scutellarin amount (μ g)	Conversion back (μ g)	Peak area
Numbering	Scutellarin amount (μ g)	Conversion back (μ g)	Peak area	1 2 3 4 5	0.1956 0.3912 0.5868 0.7824 0.9780	0.1886 0.3771 0.5657 0.7542 0.9428	603722 1224078 1835374 2430678 3038182

Regression equation: Y=3222232.53x+3654.50

Correlation coefficient: γ=0.9999

The result shows: scutellarin is good in 0.1886 μ g～0.9428 μ g scope internal linear.

As calculated, the standard curve of scutellarin is crossed initial point, therefore selects for use one point external standard method to measure the content of scutellarin.

Accurate scutellarin reference substance solution (0.0489mg/ml) the 10 μ l that draw of 7 precision experiment inject chromatograph of liquid, measure 5 times, investigate reference substance solution precision, and measurement result sees the following form.

The precision test

Test number (TN)	1	2	3	4	5	Meansigma methods	RSD(％)
Test number (TN)	1	2	3	4	5	Meansigma methods	RSD(％)	Peak area	1572015	1570202	1566206	1559384	1557017	1564965	0.42

The result shows that reference substance solution precision is good.

8 stability experiments

8.1 accurate scutellarin reference substance solution (0.0489mg/ml) the 10 μ l that draw of the stability experiment of reference substance solution inject chromatograph of liquid, inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively.

The reference substance solution stability test

Time (h)	0	2	4	8	24	Meansigma methods	RSD(％)
Time (h)	0	2	4	8	24	Meansigma methods	RSD(％)	Peak area	1572015	1570202	1566206	1559384	1557017	1564965	0.42

The result shows that 24 hours internal stabilities of reference substance solution are good.

8.2 the accurate need testing solution 10 μ l that draw of the stability experiment of need testing solution inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively.

The need testing solution stability experiment

Time (h)	0	2	4	8	24	Average	RSD
Time (h)	0	2	4	8	24	Average	RSD	Content (mg/ bottle)	2.361	2.354	2.371	2.336	2.328	2.350	0.75

The result shows that 24 hours internal stabilities of need testing solution are good.

9 repeated experiments are got this product, by operating under the preparation of text need testing solution and the algoscopy item.

Repeated experiment

Numbering	1	2	3	4	5	Meansigma methods	RSD(％)
Numbering	1	2	3	4	5	Meansigma methods	RSD(％)	Content (mg/ bottle)	2.294	2.402	2.358	2.377	2.384	2.363	1.76

The application of sample absorption method is adopted in the experiment of 10 average recoveries, get this product under the content uniformity item, get content, mixing is therefrom got about 0.13g (totally 6 parts), the accurate title, decide, split in the 25ml measuring bottle, accurate respectively scutellarin reference substance solution (C=0.626mg/ml) 1.0ml that adds adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min, take out, put, add methanol to scale to room temperature, shake up, filter with microporous filter membrane (0.45 μ m), the accurate subsequent filtrate 10 μ l that draw inject chromatograph of liquid, measure, promptly.

The content of scutellarin is in the compound Chinese medicinal preparation: 4.643mg/g.

The average recovery experiment

Numbering	Test sample sample weighting amount (g)	Pure product amount (mg) in the test sample	Scutellarin addition (mg)	Conversion back (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample sample weighting amount (g)	Pure product amount (mg) in the test sample	Scutellarin addition (mg)	Conversion back (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5 6	0.12605 0.13027 0.12281 0.12595 0.12304 0.12601	0.5853 0.6048 0.5702 0.5848 0.5713 0.5851	0.626 0.626 0.626 0.626 0.626 0.626	0.6035 0.6035 0.6035 0.6035 0.6035 0.6035	1.182 1.190 1.158 1.177 1.154 1.175	98.81 96.94 97.35 98.12 96.49 97.72

Average recovery rate=97.57%, RSD=0.85%

11 sample sizes are measured and are got this product three batch samples, according to the preparation of text need testing solution and the operation under the algoscopy item.

The scutellarin assay

Lot number	Scutellarin content (mg/ bottle)
Lot number	Scutellarin content (mg/ bottle)	1	2.331
2	2.364	1	2.331
2	2.364	3	2.278

Determination of total flavonoids in experimental example 13 compound Chinese medicinal preparation

1 instrument and reagent

(1) key instrument:

Ultraviolet spectrophotometer TU-1810SPC Beijing Puxi General Instrument Co., Ltd

Electronic analytical balance BP211D SARTORIUS

Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.

(2) reagent:

Rutin Nat'l Pharmaceutical ﹠ Biological Products Control Institute (lot number: 0080-9705)

Ethanol analytical pure atropic is Fine Chemical Co., Ltd now

Aluminum nitrate analytical pure Tianjin chemical reagent three factories

Sodium nitrite analytical pure Tianjin chemical reagent three factories

Northization glass purchase and sale center, sodium hydroxide analytical pure Tianjin

Control substance of Rutin is got in 2 selections that detect wavelength, operates by the preparation method of text reference substance solution, obtains reference substance solution.Get compound Chinese medicinal preparation, operate, obtain need testing solution by the preparation method of need testing solution in the text algoscopy.Draw control substance of Rutin solution, need testing solution, press the text total flavones and measure item method suggested down, in 400～800nm wave-length coverage, scan, the result shows, reference substance solution and need testing solution all have absorption maximum at 510nm, and solvent is noiseless, therefore select the detection wavelength of 510nm as general flavone content in the spectrophotometry compound Chinese medicinal preparation.

The preparation precision of 3 reference substance solution takes by weighing at the control substance of Rutin 20mg of 120 ℃ of drying under reduced pressure to constant weight, puts in the 100ml measuring bottle, adds an amount of supersound process of 60% alcoholic solution (power 250W, frequency 33KHz) makes dissolving, take out, put, add 60% alcoholic solution to scale to room temperature, shake up, precision is measured 25ml, puts in the 50ml measuring bottle, and thin up is to scale, shake up, promptly get (containing anhydrous rutin 0.1mg among every 1ml).

This product under the content uniformity item is got in the preparation of 4 need testing solutions, gets content, and mixing is got about 0.25g, put in the 25ml measuring bottle, add an amount of supersound process of 60% alcoholic solution (power 250W, frequency 33KHz) and make dissolving, take out, put to room temperature, add 60% alcoholic solution, shake up, promptly to scale.

5 algoscopy precisions are measured reference substance solution and each 3.0ml of need testing solution, split in the 10ml measuring bottle, respectively add 30% alcoholic solution and make into 5.0ml, precision adds sodium nitrite solution (1 → 20) 0.3ml and shakes up, placed 6 minutes, and added aluminum nitrate solution (1 → 10) 0.3ml again, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4.0ml adds water to scale again, shakes up, placed 15 minutes, with the retinue solvent is blank, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 B), measures trap respectively at the wavelength place of 510nm, calculate, promptly.

The preparation precision of 6 standard curves is measured control substance of Rutin solution (C=0.1064mg/ml) 0.0ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, split in the 10ml measuring bottle, add 30% alcoholic solution respectively and make into 5.0ml, precision adds sodium nitrite solution (1 → 20) 0.3ml, shake up, placed 6 minutes, add aluminum nitrate solution (1 → 10) 0.3ml again, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4.0ml adds water to scale again, shake up, placing 15 minutes, is blank with the retinue solvent, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 B), measure trap at 510nm wavelength place, with the trap is vertical coordinate, and concentration (mg/ml) is figure for abscissa, the drawing standard curve.

Total flavones standard curve determination result

Numbering	Rutin concentration (mg/ml)	Trap
Numbering	Rutin concentration (mg/ml)	Trap	1 2 3 4 5 6	0.000 0.0110 0.0210 0.0320 0.0430 0.0530	0.000 0.119 0.226 0.343 0.468 0.572

Regression equation: Y=lO.818x-0.0005:

Correlation coefficient: γ=0.9999;

The result shows: rutin is good in 0.0110～0.0530mg/ml scope internal linear.

As calculated, the standard curve of rutin is crossed initial point, therefore selects for use one point external standard method to measure content of total flavone.

The accurate absorption of 7 precision test control substance of Rutin solution (concentration: 0.1064mg/ml) 3.0ml, put in the 10ml measuring bottle, press the method under the text algoscopy item, from " add 30% alcoholic solution and make into 5.0ml ", operation is in accordance with the law measured trap at the wavelength place of 510nm, measures 5 times.

The precision test

Numbering	1	2	3	4	5	Average	RSD(％)
Numbering	1	2	3	4	5	Average	RSD(％)	Trap	0.343	0.343	0.342	0.340	0.337	0.341	0.75

The result shows that reference substance solution precision is good.

8 stability tests

8.1 the accurate control substance of Rutin solution (concentration: 0.1064mg/ml) 3.0ml of drawing of reference substance solution stability test, put in the 10ml measuring bottle, press the method under the text algoscopy item, from " add 30% alcoholic solution and make into 5.0ml ", operation in accordance with the law, wavelength place at 510nm measures trap, respectively at 0,5,10,15,30 minute replication once.

The reference substance solution stability test

Time (min)	0	5	10	15	30	Average	RSD(％)
Time (min)	0	5	10	15	30	Average	RSD(％)	Trap	0.343	0.343	0.342	0.340	0.337	0.341	0.75

The result shows that reference substance solution has good stability.

8.2 the accurate need testing solution 1.0ml that draws of need testing solution stability test, put in the 10ml measuring bottle, press the method under the text algoscopy item, from " add 30% alcoholic solution and make into 5.0ml ", operation in accordance with the law, measure trap at the wavelength place of 510nm, respectively 0,5,10,15,30min measures trap.

The need testing solution stability test

Time (min)	0	5	10	15	30	Average	RSD(％)
Time (min)	0	5	10	15	30	Average	RSD(％)	Content (mg/ bottle)	8.015	8.204	8.118	8.095	8.003	8.087	1.02

The result shows that need testing solution is good at the 30min internal stability.

9 replica tests are got this product, by operating under the preparation of text need testing solution and the algoscopy item.

Replica test

Time (min)	0	5	10	15	30	Average	RSD(％)
Time (min)	0	5	10	15	30	Average	RSD(％)	Content (mg/ bottle)	8.027	8.084	8.002	8.125	8.093	8.066	0.62

The result shows that repeatability is good.

The application of sample absorption method is adopted in the test of 10 average recoveries, gets this product under the content uniformity item, gets content, mixing is got 0.13g (totally 6 parts), and accurate the title decides, split in the 25ml measuring bottle, precision takes by weighing through the control substance of Rutin 30.24mg of 120 ℃ of drying under reduced pressure to constant weight, puts in the 25ml measuring bottle, add an amount of supersound process of 60% ethanol and make dissolving, take out, put to room temperature, fixed with 60% ethanol to scale, shake up, precision is measured 1.5ml (totally 6 parts), splits in the above-mentioned 25ml measuring bottle, adds an amount of supersound process of 60% alcoholic solution (power 250W, frequency 33KHz) makes dissolving, take out, put, be diluted to scale with 60% alcoholic solution to room temperature, shake up, filter, precision is measured subsequent filtrate 3.0ml, splits in the 10ml measuring bottle, according to the method under the text algoscopy item, from " add 30% alcoholic solution and make into 5.0ml ", operation is in accordance with the law measured trap at the wavelength place of 510nm, calculate, promptly.

Content of total flavone is in the compound Chinese medicinal preparation: 15.85mg/g.

The average recovery test

Numbering	Test sample sample weighting amount (g)	General flavone content in the test sample (mg)	Rutin addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample sample weighting amount (g)	General flavone content in the test sample (mg)	Rutin addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5 6	0.12227 0.13051 0.12887 0.13056 0.12407 0.12595	1.9380 2.0686 2.0426 2.0694 1.9665 1.9963	1.8144 1.8144 1.8144 1.8144 1.8144 1.8144	3.688 3.842 3.823 3.847 3.745 3.793	96.46 97.73 98.12 97.95 98.03 99.02

Average recovery rate=97.89%, RSD=0.85%.

The assay of 11 samples is got this product three batch samples, according to the preparation of text need testing solution and the operation under the algoscopy item, working sample content.

Content of total flavone is measured

Lot number	General flavone content (mg/ bottle)
Lot number	General flavone content (mg/ bottle)	1 2 3	8.124 8.073 8.223

Total saponin content is measured in experimental example 14 compound Chinese medicinal preparation

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)	13.46	13.57	13.28	13.39	13.05	13.35	1.48

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)	13.66	13.21	13.34	13.46	13.82	13.50	1.81

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 (0.662mg/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: 26.53mg/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	25.37 22.64 25.08 24.79 23.04	0.6731 0.6006 0.6654 0.6577 0.6113	0.662 0.662 0.662 0.662 0.662	1.327 1.245 1.306 1.309 1.255	98.81 97.32 96.78 98.41 97.21

Average recovery rate=97.71% RSD=0.88%

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	13.32 14.07 13.59

Panax Notoginseng saponin R in experimental example 15 compound Chinese medicinal preparation 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 50ml 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, makes negative test sample by this product preparation technology.Precision takes by weighing 2.40572g, 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 is therefrom got about 2.5g, and accurate the title decides, and 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)	0.6574 3.677 4.187	0.6631 3.525 4.192	0.6702 3.649 4.306	0.6592 3.594 4.244	0.6581 3.628 4.259	0.6616 3.615 4.238	0.80 1.62 1.17

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)	0.6601 3.602 4.178	0.6705 3.713 4.125	0.6693 3.695 4.213	0.6901 3.688 4.306	0.6825 3.592 4.278	0.6745 3.658 4.220	1.75 1.55 1.74

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 50ml 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: 0.3784mg/ml, 1.4904mg/ml, 2.2136mg/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 2.325,7.187 respectively, 8.292mg/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	1.23044 1.26051 1.24769 1.25308 1.24995	1.6303 1.6702 1.6532 1.6603 1.6562	1.5136 1.5136 1.5136 1.5136 1.5136	3.110 3.154 3.154 3.152 3.139	97.74 98.02 99.13 98.56 97.95

Average recovery rate=98.28%; RSD=0.57%.

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	1.23044 1.26051 1.24769 1.25308 1.24995	8.8432 9.0593 8.9671 9.0059 8.9834	5.9616 5.9616 5.9616 5.9616 5.9616	14.604 14.775 14.692 14.796 14.853	96.64 95.87 96.03 97.12 98.45

Average recovery rate=96.82%; RSD=1.07%.

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	1.23044 1.26051 1.24769 1.25308 1.24995	10.2028 10.4521 10.3458 10.3905 10.3646	8.8544 8.8544 8.8544 8.8544 8.8544	18.740 19.134 18.976 18.972 19.107	96.42 98.05 97.47 96.92 98.74

Average recovery rate=97.52%; RSD=0.94%.

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	0.6491 0.6762 0.6639	3.552 3.649 3.497	4.185 4.203 4.229	8.386 8.528 8.390

Experimental example 16 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 1100series 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, all purchase 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: Platinum C18 4.6 * 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 (O.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 3111269 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)	0.03371	0.03242	0.03259	0.03284	0.03207	0.03273	1.89

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)	0.03223	0.03196	0.03205	0.03182	0.03228	0.03207	0.59

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 (0.1224mg/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 0.06301mg/g in the known drug compositions after measured.

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	2.48257 2.49036 2.47904 2.48884 2.50471 2.47906	0.1564 0.1569 0.1562 0.1568 0.1578 0.1562	0.1224 0.1224 0.1224 0.1224 0.1224 0.1224	0.2757 0.2770 0.2774 0.2775 0.2774 0.2777	97.49 98.12 99.03 98.59 97.73 99.24

Astragaloside average recovery rate=98.37%; RSD=0.72%

Test agent was three batches during 10 3 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	0.03273 0.03057 0.03346

Formononetin, hair stamen formononetin assay in experimental example 17 compound Chinese medicinal preparation

1 instrument and reagent

1.1 key instrument:

High performance liquid chromatograph LC-2010AHT SHIMADZU

Electronic analytical balance BP211D SARTORIUS

Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.

1.2 reagent:

Methanol analytical pure Beijing Chemical Plant

Acetonitrile chromatographically pure Di Ma company

The pure Beijing of phosphate analysis chemical reagents corporation

2 formononetin, hair stamen formononetin provide formononetin, hair stamen formononetin by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, measure purity all greater than 98% through high performance liquid chromatography (normalization method), meet assay reference substance requirement.

Formononetin is got in the selections of 3 detection wavelength, hair stamen formononetin reference substance is an amount of, and accurate the title decides, and branch adds methanol and makes the solution that every 1ml contains about 0.05mg, in the interscan of 190～400nm wave-length coverage.The result shows that formononetin, hair stamen formononetin all have absorption maximum at the 250nm place, selects the detection wavelength of 250nm as formononetin, hair stamen formononetin assay.

4 chromatographic conditions

Chromatograph: SHIMADZU LC-2010AHT;

Chromatographic column: Diamonsil ODS 250mm * 4.6mm 5 μ m;

Mobile phase: methanol-water is a mobile phase, gradient elution, and solvent ratios is from 0 minute to 10 minutes, and the ratio of water is for rising to 50% from 40%, and from 10 minutes to 30 minutes, the ratio of water was for rising to 60% from 50%, and from 30 minutes to 50 minutes, the ratio of water was 60%;

Flow velocity: 1.0ml/min;

Column temperature: 30 ℃;

Sample size: 10 μ l;

Detect wavelength: 250nm.

The preparation precision of reference substance solution takes by weighing formononetin, hair stamen formononetin reference substance is an amount of, adds methanol and makes the solution that every 1ml contains 0.005mg, promptly.

This product under the content uniformity item is got in the preparation of need testing solution, gets content, mixing, therefrom get about 0.5g, the accurate title, decide, and puts in the 5ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m).

Obtain formononetin, hair stamen formononetin reference substance, test sample chromatogram according to above-mentioned chromatographic condition, its number of theoretical plate n is all greater than 2000, formononetin, hair stamen formononetin chromatographic peak separate clear complete with close peak, separating degree is all greater than 1.5, and solvent is noiseless.

5 linear relationships investigation precision is measured formononetin, hair stamen formononetin reference substance mixed solution (every 1ml contains formononetin 0.04808mg, hair stamen formononetin 0.04912mg respectively) 0.4,0.8,1.2,1.6,2.0ml, split in the 10ml measuring bottle, be diluted to scale with methanol, shake up, the therefrom accurate respectively 10 μ l that draw, inject chromatograph of liquid, measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).Be respectively vertical coordinate with the peak area, sample size (μ g) is abscissa mapping, drawing standard curve.

Formononetin standard curve determination result

Numbering	Formononetin amount (μ g)	Peak area
Numbering	Formononetin amount (μ g)	Peak area	1 2 3 4 5	0.019232 0.038464 0.057696 0.076928 0.09616	120005 241083 360061 481146 600924

Regression equation: Y=6249485.23x+73.50

Correlation coefficient: γ=0.9999

The result shows: formononetin is good in 0.019232 μ g～0.09616 μ g scope internal linear.

Hair stamen formononetin standard curve determination result

Numbering	Hair stamen formononetin amount (μ g)	Peak area
Numbering	Hair stamen formononetin amount (μ g)	Peak area	1 2 3 4 5	0.019648 0.039296 0.058944 0.078592 0.09824	102151 203094 305887 407377 509362

Regression equation: Y=5184777.08x-37.30

Correlation coefficient: γ=0.9999

The result shows: hair stamen formononetin is good in 0.019648 μ g～0.09824 μ g scope internal linear.

As calculated, the standard curve of formononetin, hair stamen formononetin is all crossed initial point, therefore selects for use one point external standard method to measure the content of formononetin, hair stamen formononetin.

Accurate formononetin, hair stamen formononetin reference substance mixed solution (every 1ml contains formononetin 0.004808mg, hair stamen formononetin 0.004912mg respectively) the 10 μ l of drawing of 6 precision experiment, inject chromatograph of liquid, measure 5 times, investigate reference substance solution precision, measurement result sees the following form.

The precision test

Test number (TN)	1	2	3	4	5	Meansigma methods	RSD(％)
Test number (TN)	1	2	3	4	5	Meansigma methods	RSD(％)	Formononetin hair stamen formononetin	296784 255603	301152 267796	299716 255873	302058 257601	298749 259714	299692 258117	0.69 1.02

The result shows that reference substance solution precision is good.

7 stability experiments

7.1 accurate formononetin, hair stamen formononetin reference substance mixed solution (every 1ml contains formononetin 0.004808mg, hair stamen formononetin 0.004912mg respectively) the 10 μ l of drawing of the stability experiment of reference substance solution, inject chromatograph of liquid, inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively.

The reference substance solution stability test

Time (h)	0	2	4	8	24	Meansigma methods	RSD(％)
Time (h)	0	2	4	8	24	Meansigma methods	RSD(％)	Formononetin hair stamen formononetin	296784 255603	301152 267796	299716 255873	302058 257601	298749 259714	299692 258117	0.69 1.02

7.2 the accurate need testing solution 10 μ l that draw of the stability experiment of need testing solution inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively.

The need testing solution stability experiment

Time (h)	0	2	4	8	24	Average	RSD％
Time (h)	0	2	4	8	24	Average	RSD％	Formononetin (mg/ bottle) hair stamen formononetin (mg/ bottle)	0.02216 0.02415	0.02195 0.02396	0.02137 0.02387	0.02224 0.02402	0.02208 0.02375	0.02196 0.02395	1.58 0.63

8 repeated experiments are got this product, by operating under the preparation of text need testing solution and the algoscopy item.

Repeated experiment

Numbering	1	2	3	4	5	Average	RSD％
Numbering	1	2	3	4	5	Average	RSD％	Formononetin (mg/ bottle) hair stamen formononetin (mg/ bottle)	0.02207 0.02365	0.02195 0.02402	0.02236 0.02337	0.02148 0.02411	0.02215 0.02396	0.02200 0.02382	1.49 1.29

The application of sample absorption method is adopted in the experiment of 9 average recoveries, gets this product under the content uniformity item, gets content, and mixing is therefrom got about 0.25g, and accurate the title decides, and puts in the 5ml measuring bottle; Precision is measured formononetin, hair stamen formononetin reference substance mixed solution (every 1ml contains formononetin 0.00956mg, hair stamen formononetin 0.00972mg respectively) 1ml (totally 5 parts), splits in the above-mentioned 5ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min, take out, put to room temperature, it is fixed to scale to add methanol, shake up, filter with microporous filter membrane (0.45 μ m), the accurate subsequent filtrate 10 μ l that draw inject chromatograph of liquid, measure, promptly.

The content of formononetin is in the compound Chinese medicinal preparation: 0.04323mg/g; The content of hair stamen formononetin is: 0.04680mg/g

The experiment of formononetin average recovery

Numbering	Test sample sample weighting amount (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample sample weighting amount (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	0.24484 0.25023 0.24961 0.25703 0.24485	0.0106 0.0108 0.0108 0.0111 0.0106	0.00956 0.00956 0.00956 0.00956 0.00956	0.02001 0.02014 0.02002 0.02056 0.01989	98.58 97.54 96.49 98.85 97.36

Average recovery rate=97.76%, RSD=0.98%

The experiment of hair stamen formononetin average recovery

Numbering	Test sample sample weighting amount (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample sample weighting amount (g)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	0.24484 0.25023 0.24961 0.25703 0.24485	0.0115 0.0117 0.0117 0.0120 0.0115	0.00972 0.00972 0.00972 0.00972 0.00972	0.02090 0.02112 0.02129 0.02155 0.02089	97.15 96.83 98.82 97.95 97.04

Average recovery rate=97.56%, RSD=0.84%

10 sample sizes are measured and are got this product three batch samples, according to the preparation of text need testing solution and the operation under the algoscopy item.

Assay

Lot number	Formononetin (mg/ bottle)	Hair stamen formononetin (mg/ bottle)
Lot number	Formononetin (mg/ bottle)	Hair stamen formononetin (mg/ bottle)	1 2 3	0.02203 0.02196 0.02304	0.02274 0.02195 0.02208

The specific embodiment

Embodiment 1: adopt liquid chromatography for measuring to be characterized as main finger printing with Herba Erigerontis, Radix Astragali flavone constituents

(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds water and make the solution that every 1ml contains 1mg, shakes up, promptly;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is 80% acetonitrile, and Mobile phase B is 0.1% phosphoric acid solution, gradient elution, and solvent ratios was from 0 minute to 80 minutes, and the ratio of mobile phase A rises to 70% by 15%, and the ratio of Mobile phase B rises to 30% by 85%; Flow velocity is 1.0ml/min, and the detection wavelength is 230 ± 2nm, and column temperature is 40 ℃;

(4) formulation of standard finger-print: the means of testing that is characterized as main standard finger-print with said method as formulation with Herba Erigerontis, Radix Astragali flavone constituents; Accurate respectively object of reference solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, according to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, retention time with definite object of reference chromatographic peak is a benchmark, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 24;

(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the freeze-dried powder to be measured with Herba Erigerontis, Radix Astragali flavone constituents, prepare the finger printing of freeze-dried powder to be measured;

(6) with the finger printing and the contrast of above-mentioned standard finger-print of freeze-dried powder to be measured, meet the following requirements:

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

II. in the freeze-dried powder 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%.

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: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds methanol and make the solution that every 1ml contains 50mg, shakes up, promptly;

(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 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 15;

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

Embodiment 3: adopt liquid chromatography for measuring to be characterized as main finger printing with Herba Erigerontis, Radix Astragali flavone constituents

(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder, adds methanol and make the solution that every 1ml contains 1mg, shakes up, promptly;

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing scutellarin, adds ethanol and make the solution that every 1ml contains 0.1mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is 80% acetonitrile, and Mobile phase B is 1% glacial acetic acid solution, gradient elution, and solvent ratios was from 0 minute to 80 minutes, and the ratio of mobile phase A rises to 70% by 15%, and the ratio of Mobile phase B rises to 30% by 85%; Flow velocity is 1.0ml/min, and the detection wavelength is 230 ± 2nm, and column temperature is 40 ℃;

(4) formulation of standard finger-print: the means of testing that is characterized as main standard finger-print with said method as formulation with Herba Erigerontis, Radix Astragali flavone constituents; Accurate respectively object of reference solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, according to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, retention time with definite object of reference chromatographic peak is a benchmark, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 26;

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 that precision takes by weighing freeze-dried powder to be measured, adds ethanol and make the solution that every 1ml contains 50mg, shakes up, promptly;

(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg ₁In right amount, add ethanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-1% glacial acetic acid, gradient elution, solvent ratios is from 0 minute to 14 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 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 14;

(5) with the described method in (1)～(3) as in the freeze-dried powder to be measured based on the means of testing of the finger printing of Radix Notoginseng composition characteristics, prepare the finger printing of freeze-dried powder to be measured;

Embodiment 5: adopt liquid chromatography for measuring to be characterized as main finger printing with Herba Erigerontis, Radix Astragali flavone constituents

(1) preparation of need testing solution: precision is measured injection 1ml, puts in the 50ml measuring bottle, is diluted with water to scale, shakes up, promptly;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is a methanol, and Mobile phase B is 0.2% formic acid solution, gradient elution, and solvent ratios was from 0 minute to 80 minutes, and the ratio of mobile phase A rises to 65% by 18%, and the ratio of Mobile phase B reduces to 35% by 82%; Flow velocity is 1.0ml/min, and the detection wavelength is 230 ± 2nm, and column temperature is 35 ℃;

(4) formulation of standard finger-print: the means of testing that is characterized as main standard finger-print with said method as formulation with Herba Erigerontis, Radix Astragali flavone constituents; Accurate respectively object of reference solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, according to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, retention time with definite object of reference chromatographic peak is a benchmark, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 25;

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

(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, meet the following requirements:

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

II. in the injection pin finger printing to be measured, non-total peak area must not surpass 5% 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: get injection as need testing solution:

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is methanol-0.1% phosphoric acid, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of methanol is 20%, and from 10 minutes to 40 minutes, the ratio of methanol was for to rise to 40% from 20%, from 45 minutes to 60 minutes, the ratio of methanol is for rising to 80% from 400%, and flow velocity is 1ml/min, detect wavelength 203 ± 2nm, 40 ℃ 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 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 13;

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

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

Embodiment 7: the thin layer chromatography discrimination method of Herba Erigerontis medical material, scutellarin in the freeze-dried powder

Get freeze-dried powder 1g to be measured, add methanol 10ml and extract, centrifugal, get supernatant as need testing solution; The preparation of Herba Erigerontis control medicinal material solution: get the Herba Erigerontis control medicinal material, add ethyl acetate extraction, filter, filtrate volatilizes, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the scutellarin reference substance, add methanol and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, put on same polyamide membrane with strip tape respectively, be developing solvent with methanol-ethyl acetate-formic acid at 7: 2: 1, launches, take out, dry, spray is with 2% ferric chloride alcoholic solution, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the streak of same color.

Embodiment 8: the liquid chromatograph discrimination method of scutellarin in the freeze-dried powder

Get freeze-dried powder 0.25g to be measured, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution is a mobile phase at 50%: 50%, and the detection wavelength is 335nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 9: Radix Notoginseng, ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The thin layer chromatography discrimination method

Get freeze-dried powder 5g to be measured, extract with n-butyl alcohol 20ml, filter, filtrate volatilizes, and residue adds methanol 5ml dissolving, as need testing 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 ₁Reference substance adds methanol respectively and makes 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, 105 ℃ are dried by the fire to 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.

Embodiment 10: ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Liquid chromatograph differentiate

Get freeze-dried powder 2.5g to be measured, put in the 50ml measuring bottle, add methanol 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, take out, put, add methanol to scale to room temperature, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The methanol solution of reference substance is 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.

Embodiment 11: the thin layer chromatography discrimination method of the Radix Astragali in the freeze-dried powder

Get middle freeze-dried powder 5g to be measured, add ethanol 20ml and extract, filter the filtrate evaporate to dryness, residue adds 0.3% sodium hydroxide solution 10ml dissolving, filter, filtrate is extracted with ethyl acetate 20ml with hydrochloric acid condition pH value to 5～6, filter, filtrate evaporate to dryness, residue add ethyl acetate 1ml 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 12: the thin layer chromatography discrimination method of astragaloside in the freeze-dried powder

Get freeze-dried powder 5g to be measured, be added on the neutral alumina post after adding methanol 10ml dissolving, with 40% methanol 40ml eluting, collect eluent, evaporate to dryness, residue add water-saturated n-butanol extraction 3 times after adding water 10ml dissolving, each 10ml, with and n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue is with methanol 2ml dissolving, 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 13: the liquid chromatograph discrimination method of astragaloside in the freeze-dried powder

Get freeze-dried powder 5g to be measured, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml, with and n-butyl alcohol liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 40% ethanol 30ml, 70% ethanol 80ml eluting successively, collect 70% ethanol elution part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, 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 14: the thin layer chromatography discrimination method of formononetin, hair stamen formononetin in the freeze-dried powder

Get freeze-dried powder 3g to be measured, add methanol 10ml and extract, extracting solution is as need testing solution; Other gets formononetin, hair stamen formononetin 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 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.

Embodiment 15: the liquid chromatograph discrimination method of formononetin, hair stamen formononetin in the freeze-dried powder

Get freeze-dried powder 0.5g to be measured, the accurate title, decide, and puts in the 5ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); 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.

Embodiment 16: the assay of scutellarin in the freeze-dried powder

Get freeze-dried powder 0.25g to be measured, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution 50%:50% is a mobile phase, and the detection wavelength is 335nm; A bit calculate with external standard, said preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg.

Embodiment 17: content of total flavone is measured in the freeze-dried powder

Get freeze-dried powder 0.25g to be measured, put in the 25ml measuring bottle, add an amount of supersound process of 60% alcoholic solution (power 250W, frequency 33KHz) makes dissolving, take out, put, add 60% alcoholic solution to scale to room temperature, shake up, precision is measured 3ml, puts in the 50ml measuring bottle, adds methanol to scale, shake up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at the wavelength place of 335nm, calculate with one point external standard method, said preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the limit of total flavones in scutellarin, must not be less than 10mg.

Embodiment 18: ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In assay

Get freeze-dried powder 2.5g to be measured, put in the 50ml measuring bottle, add methanol 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, take out, put, add methanol to scale to room temperature, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The methanol solution of middle reference substance is 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, square preparation to be measured is unit quantity to be equivalent to every day with output, and content limit should be following 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 freeze-dried powder

Get freeze-dried powder 50mg to be measured, 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, shake up to scale, precision is measured 1ml, put in the 25ml measuring bottle, water bath method takes out immediately, 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, shakes up immediately, 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, 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 freeze-dried powder

Get freeze-dried powder 5g to be measured, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml, with and n-butyl alcohol liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 40% ethanol 30ml, 70% ethanol 80ml eluting successively, collect 70% ethanol elution part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, 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, said 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.

Embodiment 21: the assay of formononetin, hair stamen formononetin in the freeze-dried powder

Get freeze-dried powder 0.5g to be measured, the accurate title, decide, and puts in the 5ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); 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 30 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, compound Chinese medicinal preparation to be measured is unit quantity to be equivalent to every day with output, and content limit should be following two:

Embodiment 22: the thin layer chromatography discrimination method of Herba Erigerontis medical material, scutellarin in the injection

Get injection 10ml to be measured, evaporate to dryness, residue add methanol 1ml dissolving, as need testing solution; Get the Herba Erigerontis control medicinal material, add ethyl acetate extraction, filter, filtrate volatilizes, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the scutellarin reference substance, add methanol and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, respectively putting on same silica gel g thin-layer plate, with benzene-ethyl acetate-formic acid-water 7: 2: 1: 0.2 is developing solvent, launches, take out, dry, spray is with 1% aluminum chloride alcoholic solution, 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.

Embodiment 23: the liquid chromatograph discrimination method of scutellarin in the injection

Get injection 5ml to be measured, put in the 25ml measuring bottle, add water and decide to shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m) to scale; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-1% glacial acetic acid aqueous solution is a mobile phase at 45%: 55%, and the detection wavelength is 335nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 24: Radix Notoginseng, ginsenoside Rg in the injection ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The thin layer chromatography discrimination method

Get injection 20ml to be measured, evaporate to dryness, residue methanol 5ml is as need testing 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 methylene chloride discards dichloromethane solution, 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 ₁Reference substance adds methanol respectively and makes 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 H lamellae, with n-butyl alcohol-ethyl acetate-upper solution of 5: 1: 33 of water is developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to 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.

Embodiment 25: ginsenoside Rg in the injection ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Liquid chromatograph differentiate

Get injection 25ml to be measured, put in the 50ml measuring bottle, add water to scale, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The methanol solution of middle reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-1% glacial acetic acid aqueous solution 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 35 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 26: the thin layer chromatography discrimination method of the Radix Astragali in the injection

Get injection 20ml to be measured, evaporate to dryness, residue add 0.3% sodium hydroxide solution 10ml dissolving, filter, and filtrate is extracted with n-butyl alcohol 20ml with hydrochloric acid condition pH value to 5～6, filter, and filtrate evaporate to dryness, residue add methanol 1ml 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 methylene chloride-methanol at 5: 0.8, 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 27: the thin layer chromatography discrimination method of astragaloside in the injection

Get injection 20ml to be measured, evaporate to dryness, residue are added on the neutral alumina post after adding methanol 5ml dissolving, with 40% methanol 40ml eluting, collect eluent, evaporate to dryness, residue adds water-saturated n-butanol extraction 3 times after adding water 10ml dissolving, 10ml and also n-butyl alcohol liquid at every turn wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue is with methanol 2ml dissolving, 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 dichloromethane-ethanol-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 28: the liquid chromatograph discrimination method of astragaloside in the injection

Get injection 20ml to be measured, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml, with and n-butyl alcohol liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 30% methanol 30ml, 65% methanol 80ml eluting successively, collect 65% methanol-eluted fractions part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, 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-0.5% glacial acetic acid was a mobile phase in 30%: 70%, 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 29: the thin layer chromatography discrimination method of formononetin, hair stamen formononetin in the injection

Get injection 30ml to be measured, evaporate to dryness, residue add methanol 10ml and extract, and extracting solution is as need testing solution; Other gets reference substance in formononetin, the hair stamen formononetin, 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 dichloromethane-ethanol-ethyl acetate-water 3: 4: 3: 0.5 was developing solvent, launches, take out, dry, 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.

Embodiment 30: the liquid chromatograph discrimination method of formononetin, hair stamen formononetin in the injection

Get injection 10ml to be measured, evaporate to dryness, residue makes dissolving in right amount with methanol and quantitatively is transferred in the 5ml measuring bottle, adds methanol and decides to shake up to scale, filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate 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 an acetonitrile, 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 55% from 45%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 65% from 55%, from 30 minutes to 50 minutes, the ratio of water is 65%, and the detection wavelength is 250nm, 40 ℃ 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 31: the assay of scutellarin in the injection

Get injection 30ml to be measured, evaporate to dryness, residue add methanol makes dissolving also quantitatively be transferred in the 10ml measuring bottle in right amount, adds methanol and decides to shake up to scale, filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate as need testing solution; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and-1% glacial acetic acid aqueous solution is a mobile phase at 40%: 60%, and the detection wavelength is 335nm; A bit calculate with external standard, said preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg.

Embodiment 32: content of total flavone is measured in the injection

Precision is measured injection 2.5ml to be measured, puts in the 50ml measuring bottle, adds ethanol to scale, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at the wavelength place of 335nm, calculate with one point external standard method, said preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the limit of total flavones in scutellarin, must not be less than 10mg.

Embodiment 33: ginsenoside Rg in the injection ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Assay

Get injection 25ml to be measured, put in the 50ml measuring bottle, add water to scale, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-1% glacial acetic acid aqueous solution 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, injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Embodiment 34: the assay of total saponins in the injection

Precision is measured injection 0.8ml to be measured, puts in the 25ml measuring bottle water bath method, take out immediately, precision adds 5% vanillin-glacial acetic acid solution 1ml, perchloric acid 4ml, shake up, heating is 7 minutes in 60 ℃ of water-baths, takes out, immediately with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale, 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, this injection 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 35: the assay of astragaloside in the injection

Get injection 20ml to be measured, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml, with and n-butyl alcohol liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 30% methanol 30ml, 65% methanol 80ml eluting successively, collect 65% methanol-eluted fractions part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, 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-0.5% glacial acetic acid was a mobile phase in 30%: 70%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, said 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.

Embodiment 36: the assay of formononetin, hair stamen formononetin in the injection

Get injection 10ml to be measured, evaporate to dryness, residue makes dissolving in right amount with methanol and quantitatively is transferred in the 5ml measuring bottle, adds methanol and decides to shake up to scale, filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate 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 an acetonitrile, and Mobile phase B is a water, and gradient Shen is taken off, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 55% from 45%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 65% from 55%, from 30 minutes to 50 minutes, the ratio of water is 65%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be following two:

Embodiment 37: the thin layer chromatography discrimination method in the tablet in Herba Erigerontis medical material, the scutellarin

Get tablet to be measured, pulverize, therefrom get about 1g, add ethyl acetate 10ml and extract, centrifugal, supernatant evaporate to dryness, residue add methanol 5ml dissolving as need testing solution; The preparation of Herba Erigerontis control medicinal material solution: get the Herba Erigerontis control medicinal material, add n-butanol extraction, filter, filtrate volatilizes, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the scutellarin reference substance, add methanol and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, respectively putting on same silica gel H lamellae, with toluene-ethyl acetate-formic acid-water 7: 2: 1:: 0.5 is developing solvent, launches, take out, dry, spray is with 1% aluminum chloride alcoholic solution, 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.

Embodiment 38: the liquid chromatograph discrimination method of scutellarin in the tablet

Get this product 0.5g, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of mobile phase (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add mobile phase, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.5% aqueous formic acid is a mobile phase at 45%: 55%, and the detection wavelength is 335nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 39: Radix Notoginseng, ginsenoside Rg in the tablet ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The thin layer chromatography discrimination method

Get this product 10g, use water dissolution, filter, the saturated n-butyl alcohol 20ml of filtrate water extracts, and filters, and filtrate volatilizes, and residue adds ethanol 5ml dissolving, as need testing 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, and 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 ₁Reference substance adds methanol respectively and makes 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 formate-methanol-water 10: 35: 20: 8 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to 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.

Embodiment 40: ginsenoside Rg in the tablet ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Liquid chromatograph differentiate

Get this product 10g, put in the 50ml measuring bottle, add mobile phase 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, takes out, and puts to room temperature, adds mobile phase to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and-0.5% formic acid is mobile phase, gradient elution, solvent ratios is from 0 minute to 13 minutes, the ratio of acetonitrile is 18%, and from 13 minutes to 20 minutes, the ratio of acetonitrile rose to 42% by 18%, from 20 minutes to 25 minutes, the ratio of acetonitrile was 42%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 40 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 41: the thin layer chromatography discrimination method of the Radix Astragali in the tablet

Get this product 10g, add n-butyl alcohol 20ml and extract, filter the filtrate evaporate to dryness, residue adds 0.3% sodium hydroxide solution 10ml dissolving, filters, and filtrate is extracted with ethyl acetate 20ml with hydrochloric acid condition pH value to 5～6, filter, filtrate evaporate to dryness, residue add ethyl acetate 1ml 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-ethanol at 9: 0.5, 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 42: the thin layer chromatography discrimination method of astragaloside in the tablet

Get this product 10g, add ethanol 40ml supersound process and make dissolving, take out, filter, filtrate evaporate to dryness, residue are added on the neutral alumina post after adding methanol 5ml dissolving, with 50% ethanol 50ml eluting, collect eluent, evaporate to dryness, residue adds water-saturated n-butanol extraction 3 times after adding water 10ml dissolving, 10ml and also n-butyl alcohol liquid at every turn, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue is with methanol 2ml dissolving, 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 15: 5: 5 of methylene chloride-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 43: the liquid chromatograph discrimination method of astragaloside in the tablet

Get this product 10g, add water 20ml dissolving back ethyl acetate extraction, each 40ml, with and ethyl acetate liquid, with ammonia solution thorough washing 2 times, each 40ml, discard ammoniacal liquor, ethyl acetate liquid evaporate to dryness, residue add water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 35% ethanol 50ml, 65% ethanol 50ml eluting are collected 65% ethanol elution part successively, evaporate to dryness, residue shakes up with methanol 5ml dissolving, 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 methanol-0.5% formic acid was mobile phase in 45%: 55%, 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 44: the thin layer chromatography discrimination method of formononetin, hair stamen formononetin in the tablet

Get this product 6g, add methanol 10ml and extract, extracting solution is as need testing solution; Other gets formononetin, hair stamen formononetin 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 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with methylene chloride-methanol-ethyl acetate-water 3: 2: 5: 1 was developing solvent, launches, take out, dry, 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.

Embodiment 45: the liquid chromatograph discrimination method of formononetin, hair stamen formononetin in the tablet

Get this product 1g, the accurate title, decide, and puts in the 5ml measuring bottle, adds an amount of supersound process of mobile phase (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add mobile phase, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is an acetonitrile, Mobile phase B is 0.05% phosphate aqueous solution, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of Mobile phase B is for rising to 55% from 45%, and from 10 minutes to 20 minutes, the ratio of Mobile phase B was for to rise to 65% from 55%, from 30 minutes to 50 minutes, the ratio of Mobile phase B is 65%, and the detection wavelength is 250nm, 35 ℃ 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 46: the assay of scutellarin in the tablet

Get this product 0.5g, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of mobile phase (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add mobile phase, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.5% aqueous formic acid is a mobile phase at 45%: 55%, and the detection wavelength is 335nm; A bit calculate with external standard, said preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg.

Embodiment 47: content of total flavone is measured in the tablet:

Get this product 0.5g, put in the 25ml measuring bottle, add an amount of supersound process of methanol solution (power 250W, frequency 33KHz) and make dissolving, take out, put to room temperature, add methanol solution to scale, shake up, precision is measured 3ml, put in the 50ml measuring bottle, add methanol, shake up, as need testing solution to scale; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at the wavelength place of 335nm, calculate with one point external standard method, said preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the limit of total flavones in scutellarin, must not be less than 10mg.

Embodiment 48: ginsenoside Rg in the tablet ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁Assay

Get this product 10g, put in the 50ml measuring bottle, add mobile phase 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, takes out, and puts to room temperature, adds mobile phase to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and-0.5% formic acid is mobile phase, gradient elution, solvent ratios is from 0 minute to 13 minutes, the ratio of acetonitrile is 18%, and from 13 minutes to 20 minutes, the ratio of acetonitrile rose to 42% by 18%, from 20 minutes to 25 minutes, the ratio of acetonitrile was 42%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 40 ℃; Calculate with external standard method or standard curve method, compound Chinese medicinal preparation to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Embodiment 49: the assay of total saponins in the tablet

Get this product 0.1g, the accurate title, decide, and puts in the 25ml measuring bottle, add an amount of supersound process of ethanol (power 250W, frequency 33KHZ) and make dissolving, take out, put to room temperature, add ethanol, shake up to scale, precision is measured 1ml, put in the 25ml measuring bottle, water bath method takes out immediately, precision adds 3% vanillin-glacial acetic acid solution 1ml, perchloric acid 2.0ml shakes up, and heating is 10 minutes in 70 ℃ of water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale, shakes up immediately, 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 Chinese medicinal 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 50: the assay of astragaloside in the tablet

Get this product 10g, add water 20ml dissolving back ethyl acetate extraction, each 40ml, with and ethyl acetate liquid, with ammonia solution thorough washing 2 times, each 40ml, discard ammoniacal liquor, ethyl acetate liquid evaporate to dryness, residue add water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 35% ethanol 50ml, 65% ethanol 50ml eluting are collected 65% ethanol elution part successively, evaporate to dryness, residue shakes up with methanol 5ml dissolving, 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 methanol-0.5% formic acid was mobile phase in 45%: 55%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, said 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.

Embodiment 51: the assay of formononetin, hair stamen formononetin in the freeze-dried powder

Get this product powder 1g, the accurate title, decide, and puts in the 5ml measuring bottle, adds an amount of supersound process of mobile phase (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add mobile phase, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is an acetonitrile, Mobile phase B is 0.05% phosphate aqueous solution, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of Mobile phase B is for rising to 55% from 45%, and from 10 minutes to 20 minutes, the ratio of Mobile phase B was for to rise to 65% from 55%, from 30 minutes to 50 minutes, the ratio of Mobile phase B is 65%, and the detection wavelength is 250nm, 35 ℃ of column temperatures; Calculate with one point external standard method, compound Chinese medicinal preparation to be measured is unit quantity to be equivalent to every day with output, and content limit should be following two:

Claims

1, a kind of method of quality control of the compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng, the Radix Astragali, 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 Herba Erigerontis, Radix Notoginseng, the Radix Astragali 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 in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents with Herba Erigerontis, Radix Astragali flavone constituents:

3, according to the method for quality control of the described compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng, the Radix Astragali 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 in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents with Herba Erigerontis, Radix Astragali flavone constituents:

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

6, according to the method for quality control of the described compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng, the Radix Astragali 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 scutellarin in a, the compound Chinese medicinal preparation:

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

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

7, according to the method for quality control of the described compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng, the Radix Astragali 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 scutellarin in a, the compound Chinese medicinal preparation:

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

The assay of astragaloside in e, 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 Herba Erigerontis, Radix Notoginseng, the Radix Astragali, it is characterized in that: the assay result of described ejection preparation, calculate scutellarin, astragaloside, formononetin, calycosin, ginsenoside Rg according to percentage by weight ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁, all or part of material in the total saponins, total flavones total content account for deduction adjuvant and outer more than 25% of total solid of moisture in the preparation.