CN1939368A

CN1939368A - Quality control of Chinese medicinal compound preparation

Info

Publication number: CN1939368A
Application number: CN 200510107813
Authority: CN
Inventors: 于文风
Original assignee: Qiyuanyide Medicines Institute Beijing
Current assignee: Qiyuanyide Medicines Institute Beijing
Priority date: 2005-09-30
Filing date: 2005-09-30
Publication date: 2007-04-04

Abstract

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

Description

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

Technical field

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

Background technology

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

Summary of the invention

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

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

Scheme one:

Radix Notoginseng 1～99 weight portion Herba Erigerontis 99～1 weight portions

Scheme two:

Radix Notoginseng 1～99 weight portion Herba Erigerontis extract 30～0.1 weight portions

Scheme three:

Radix Notoginseng extract 0.1～30 weight portion Herba Erigerontis 99～1 weight portions

Scheme four:

Radix Notoginseng extract 0.1～30 weight portion Herba Erigerontis extract 30～0.1 weight portions

Scheme five:

Radix Notoginseng total arasaponins 0.1～30 weight portion Herba Erigerontis total flavones 30～0.1 weight portions

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

The present invention constitutes like this:

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

(1) finger printing test comprises based on the finger printing of Herba Erigerontis composition characteristics with based in the finger printing of Radix Notoginseng composition characteristics one or both;

(2) Herba Erigerontis control medicinal material, Radix Notoginseng control medicinal material, scutellarin, Panax Notoginseng saponin R ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁In the differential test method of all or part of composition;

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

The method of quality control of the described compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng is characterized in that: this method adopts liquid chromatography test based on the finger printing of Herba Erigerontis composition characteristics with based in the finger printing of Radix Notoginseng composition characteristics one or both:

A, employing liquid chromatography are tested the finger printing based on the Herba Erigerontis composition characteristics:

(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 scutellarin,, be settled to suitable concn, as object of reference solution with methanol or dissolve with ethanol;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 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 the 190-400nm scope, and column temperature is in 20～60 ℃ of scopes;

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

(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 Herba Erigerontis, the Radix Paeoniae Alba or Radix Paeoniae Rubra composition characteristics, the finger printing of preparation testing sample;

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

B, employing liquid chromatography are tested the finger printing based on the Radix Notoginseng composition characteristics:

(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 pseudo-ginseng, comprise the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In a kind of, water or methanol or dissolve with ethanol are settled to suitable concn, as object of reference solution;

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

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

(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;

A, adopt the finger printing of liquid chromatography for measuring based on the Herba Erigerontis composition characteristics:

(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 0.15mg, 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 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: with said method as the means of testing of formulating based on the standard finger-print of Herba Erigerontis composition characteristics; 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 3～20;

(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, the preparation testing sample finger printing;

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

(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 12 minutes, the ratio of acetonitrile is 15%, from 14 minutes to 45 minutes, the ratio of acetonitrile is for rising to 30% from 15%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was for to rise to 80% from 30%, flow velocity is 1ml/min, detection wavelength 203 ± 2nm, 30 ℃ of column temperatures;

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the finger printing of three composition characteristics; 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;

The method of quality control of the described compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng 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 one or both each 1～30 μ l of above-mentioned need testing solution and Herba Erigerontis control medicinal material solution, scutellarin reference substance solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On lamellae or the polyamide film, with 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 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 Radix Notoginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more, the preparation contrast solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adding chloroform or dichloromethane or aether backflow extracts, discard chloroform or dichloromethane or ether solution, residue adds water-saturated n-butanol or ethyl acetate extraction, extracting solution adds ammonia solution, divides and gets upper strata, evaporate to dryness, residue is with methanol or dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances, add methanol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, lower floor's solution or n-butyl alcohol-ethyl acetate or the Ethyl formate-water 1～10: 0.2～2 placed below 5～40: 10～100: 5～50: 0.2～3010 ℃ 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, negative noiseless;

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 Re, Panax Notoginseng saponin R ₁In the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution 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, negative noiseless.

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

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 Re, Panax Notoginseng saponin R ₁In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

The method of quality control of the described compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng 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 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:

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

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

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

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

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

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

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

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

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

Experimental example 1 is based on the preparation of the finger printing of Herba Erigerontis composition characteristics

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 0.1mg, 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 0.15mg, 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 based on the finger printing of Radix Notoginseng composition characteristics

A, experimental apparatus, reagent and sample:

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

B, chromatographic condition and system suitability experiment:

1. the selection of chromatographic column:

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

2. the selection of mobile phase:

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

3. detection wavelength determination:

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

4. instrument, chromatographic column and integral parameter:

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

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

5. the preparation of need testing solution:

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

6. the preparation of object of reference solution:

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

7. finger printing and technical parameter:

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	Problem
Condition	Problem	N-hexane-benzene-Ethyl formate-formic acid, (5-5-10-4) silica gel H lamellae ethyl acetate-benzene-acetic acid, (5-4-3) silica gel H lamellae ethyl acetate-benzene-formic acid, (5-4-3) silica gel g thin-layer plate chloroform-ethyl acetate-methyl alcohol, (7-2-4) silica GF254 lamellae toluene-ethyl acetate, (8-1) silica gel H lamellae methyl alcohol-ethyl acetate-formic acid, (10-5-0.5) silica gel g thin-layer plate methyl alcohol-ethyl acetate-formic acid, (7-2-1) PA membrane	Reference substance is expanded to the forward position reference substance not to be separated, feminine gender has the reference substance of interference not separate, feminine gender has the reference substance of interference not separate, feminine gender has the reference substance of interference not separate, it is clear that feminine gender has the reference substance of interference to be expanded to the forward position separation, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the 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	Problem
Condition	Problem	Methyl alcohol-0.05mol/L sodium hydrogen phosphate (80: 20) eight alkyl silane bonded silica gel acetonitriles-0.05mol/L sodium hydrogen phosphate (40: 60) octadecylsilane chemically bonded silica methyl alcohol-oxolane-0.05mol/L sodium hydrogen phosphate (20: 10: 70) octadecylsilane chemically bonded silica acetonitrile-oxolane-0.05mol/L sodium hydrogen phosphate (20: 10: 70) octadecylsilane chemically bonded silica methyl alcohol-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane chemically bonded silica acetonitrile-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane chemically bonded silica methyl alcohol-0.1% phosphate aqueous solution (50: 50) octadecylsilane chemically bonded silica	The too fast feminine gender of appearance time has the feminine gender of interference to have the slightly asymmetric feminine gender of the peak shape of interference to have the slightly asymmetric retention time of the peak shape of interference moderate; The peak is capable sharp-pointed; Symmetry, negative noiseless

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

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

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

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

Scutellarin assay in experimental example 7 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	2.607 2.594 2.611

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 6	0.0000 0.1956 0.3912 0.5868 0.7824 0.9780	0.0000 0.1886 0.3771 0.5657 0.7542 0.9428	0 603722 1224078 1835374 2505678 3038182

Regression equation: Y=3259091.50x-1830.06

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)	2.613	2.589	2.618	2.624	2.607	2.610	0.51

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)	2.615	2.624	2.681	2.579	2.646	2.629	1.44

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=0.6782mg/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: 4.958mg/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	0.12507 0.13158 0.12496 0.13075 0.12693 0.12559	0.6201 0.6524 0.6196 0.6483 0.6293 0.6227	0.6782 0.6782 0.6782 0.6782 0.6782 0.6782	0.6538 0.6538 0.6538 0.6538 0.6538 0.6538	1.258 1.297 1.267 1.290 1.268 1.254	97.55 98.64 99.03 98.21 97.76 96.59

Average recovery rate=97.96%, RSD=0.88%

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 3	2.518 2.497 2.715

Determination of total flavonoids in experimental example 8 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 (appendix VB of Chinese Pharmacopoeia version in 2000), 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 (appendix VB of Chinese Pharmacopoeia version in 2000), 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=10.818x-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)	6.428	6.323	6.304	6.412	6.409	0.375	0.90

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)	6.411	6.502	6.395	6.329	6.437	6.415	0.98

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 26.34mg 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: 12.18mg/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	0.12664 0.13507 0.12908 0.13012 0.12297 0.12663	1.5425 1.6452 1.5722 1.5849 1.4978 1.5424	1.5804 1.5804 1.5804 1.5804 1.5804 1.5804	3.104 3.186 3.099 3.136 3.063 3.085	98.81 97.49 96.58 98.15 99.03 97.64

Average recovery rate=97.95%, RSD=0.93%.

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	6.554 6.613 6.708

Total saponin content is measured in experimental example 9 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 (appendix VA of Chinese Pharmacopoeia version in 2000), 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 (appendix VA of Chinese Pharmacopoeia version in 2000), measure trap at the wavelength place of 547nm.With the trap is vertical coordinate, and the amount of astragaloside (μ g) is an abscissa, the drawing standard curve.

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

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

The ginsenoside Rg ₁The standard curve determination data

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

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

The precision experiment

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

5 stability tests

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

The reference substance solution stability experiment

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

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

The need testing solution stability experiment

Time (min)	0	10	20	40	60	Average	RSD％
Time (min)	0	10	20	40	60	Average	RSD％	Content (mg/ bottle)	12.16	12.61	12.49	12.59	12.58	12.49	1.51

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

Repeated experiment

Numbering	1	2	3	4	5	X is average	RSD％
Numbering	1	2	3	4	5	X is average	RSD％	Content (mg/ bottle)	12.84	12.91	12.72	12.94	12.84	12.85	0.66

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 (0.642mg/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: 24.41mg/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	24.71 25.94 26.88 27.19 26.55	0.6032 0.6332 0.6561 0.6637 0.6481	0.642 0.642 0.642 0.642 0.642	1.238 1.259 1.272 1.284 1.271	98.81 97.46 95.93 96.68 97.03

Average recovery rate=97.18% RSD=1.10%

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	13.34 12.96 13.15

Panax Notoginseng saponin R in experimental example 10 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 is by the ginsenoside Rg ₁The peak calculates 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, and therefrom precision takes by weighing 2.48425g,,, measure at 0,2,6,10,24 hour sample introduction respectively by operating under the preparation of text need testing solution.

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)	0.6504 3.424 4.439	0.6347 3.469 4.516	0.6449 3.451 4.489	0.6517 3.357 4.606	0.6432 3.408 4.582	0.6450 3.422 4.526	1.05 1.26 1.51

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 3.502 4.403	0.6508 3.479 4.507	0.6547 3.603 4.462	0.6493 3.554 4.443	0.6542 3.528 4.385	0.6538 3.533 4.440	0.64 1.36 1.09

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: 0.385mg/ml, 1.694mg/ml, 2.403mg/ml), add an amount of supersound process of methanol (power 250W, frequency 33KHz) and make dissolving, take out, put to room temperature, add methanol to scale, shake up, filter with microporous filter membrane (0.45 μ m), the accurate 10 μ l that draw, inject chromatograph of liquid, measure, promptly.

Panax Notoginseng saponin R in the known after measured compound Chinese medicinal preparation ₁, the ginsenoside Rg ₁And ginsenoside Rb ₁Content be 1.242,6.711 respectively, 8.434mg/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	1.22413 1.23369 1.23085 1.24004 1.23639	1.5204 1.5322 1.5287 1.5401 1.5356	1.540 1.540 1.540 1.540 1.540	3.026 3.023 3.040 3.050 3.037	97.76 96.83 98.12 98.04 97.49

Average recovery rate=97.65%; RSD=0.53%.

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	1.22413 1.23369 1.23085 1.24004 1.23639	8.2151 8.2793 8.2602 8.3219 8.2974	6.776 6.776 6.776 6.776 6.776	14.839 14.929 14.815 14.964 15.014	97.75 98.13 96.74 98.03 99.12

Average recovery rate=97.95%; RSD=0.87%.

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	1.22413 1.23369 1.23085 1.24004 1.23639	10.32 10.40 10.38 10.46 10.43	9.612 9.612 9.612 9.612 9.612	19.758 19.924 19.798 19.717 19.862	98.15 99.03 97.97 96.32 98.15

Average recovery rate=97.92%; RSD=1.01%.

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	0.6702 0.6915 0.6532	3.267 3.504 3.387	4.452 4.503 4.401	8.389 8.699 8.441

Experimental example 1 adopts the finger printing of liquid chromatography for measuring based on the Herba Erigerontis composition characteristics:

(1) preparation of need testing solution: it is an amount of that precision takes by weighing prescription powder injection formulation of the present invention, adds water and make the solution that every 1ml contains 1mg, shakes up, promptly;

(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the prescription powder injection formulation of the present invention with Herba Erigerontis, prepare the finger printing of prescription powder injection formulation of the present invention;

(6) with the finger printing of prescription powder injection formulation of the present invention 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 prescription powder injection formulation of the present invention, should be 0.90～1.00;

II. in the prescription powder injection formulation finger printing of the present invention, 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%.

Experimental example 2 adopts the finger printing of liquid chromatography test based on the Radix Notoginseng composition characteristics:

(1) preparation of need testing solution: it is an amount of that precision takes by weighing prescription powder injection formulation of the present invention, adds water and make the solution that every 1ml contains 50mg, shakes up, promptly;

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the finger printing of Radix Notoginseng composition characteristics; 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 prescription powder injection formulation of the present invention based on the means of testing of the finger printing of Radix Notoginseng composition characteristics, the finger printing of preparation testing sample;

One or more thin layer chromatography discrimination method in Herba Erigerontis medical material, the scutellarin in the experimental example 3 prescription powder injection formulations of the present invention

Get prescription powder injection formulation 2g of the present invention, add methanol 10ml and extract, centrifugal, get 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.

The liquid chromatograph discrimination method of experimental example 4 prescription powder injection formulation scutellarins of the present invention

Get prescription powder injection formulation 0.25g of the present invention, 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.

Radix Notoginseng, ginsenoside Rg in the experimental example 5 prescription powder injection formulations of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more thin layer chromatography discrimination method

Get prescription powder injection formulation 3g of the present invention, extract with n-butyl alcohol 10ml, filter, filtrate is as need testing solution; Other gets Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more, preparation control medicinal material solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adds the chloroform reflux, extract,, discards chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolves with methanol 2ml, in contrast medical material solution; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: 10 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless.

Ginsenoside Rg in the experimental example 6 prescription powder injection formulations of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more liquid chromatograph differentiate

Get prescription powder injection formulation 2.5g of the present invention, 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 Re, Panax Notoginseng saponin R ₁In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

The assay of experimental example 7 prescription powder injection formulation scutellarins of the present invention

Get prescription powder injection formulation 0.25g of the present invention, 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; 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.

Content of total flavone is measured in the experimental example 8 prescription powder injection formulations of the present invention

Prescription powder injection formulation 0.25g of the present invention puts in the 25ml measuring bottle, adds an amount of supersound process of 60% alcoholic solution (power 250W, frequency 33KHz) and makes dissolving, takes out, and puts to room temperature, adds 60% alcoholic solution 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.

Ginsenoside Rg in the experimental example 9 prescription powder injection formulations of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more assay

Get prescription powder injection formulation 2.5g of the present invention, 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 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:

(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 the experimental example 10 prescription powder injection formulations of the present invention

Get prescription powder injection formulation 50mg of the present invention, 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 2.5ml, put in the 25ml measuring bottle, water bath method takes out immediately, precision adds 5% vanillin-glacial acetic acid solution 1.25ml, perchloric acid 5ml 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, 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.

Experimental example 11 adopts the finger printing of liquid chromatography for measuring based on the Herba Erigerontis composition characteristics

(1) preparation of need testing solution: get the little hydro-acupuncture preparation of prescription of the present invention as need testing solution;

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing scutellarin, adds second and make the solution that every 1ml contains 0.15mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is 80% methanol, 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 ℃;

(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the little hydro-acupuncture preparation of prescription of the present invention with Herba Erigerontis, prepare the finger printing of the little hydro-acupuncture preparation of prescription of the present invention;

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

I. calculate the finger printing of the little hydro-acupuncture preparation of prescription of the present invention and the similarity of standard finger-print, should be 0.90～1.00;

II. in the little hydro-acupuncture preparation finger printing of prescription of the present invention, non-total peak area must not surpass 5% of total peak area;

Experimental example 12 adopts the finger printing of liquid chromatography test based on the Radix Notoginseng composition characteristics

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

(5) with the described method in (1)～(3) as in the little hydro-acupuncture preparation of prescription of the present invention based on the means of testing of the finger printing of Radix Notoginseng composition characteristics, the finger printing of preparation testing sample;

One or more thin layer chromatography discrimination method in Herba Erigerontis medical material, the scutellarin in the little hydro-acupuncture preparation of experimental example 13 prescriptions of the present invention

Get the little hydro-acupuncture preparation 10ml of prescription of the present invention, water bath method, residue add ethanol 10ml and extract, and be 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 chloroform extraction, filter, filtrate volatilizes, residue dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the scutellarin reference substance, add ethanol 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 ethanol-Ethyl formate-acetic acid at 8: 2: 2, 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.

The liquid chromatograph discrimination method of the little hydro-acupuncture preparation scutellarin of experimental example 14 prescriptions of the present invention

Get the little hydro-acupuncture preparation 10ml of prescription of the present invention, put in the 25ml measuring bottle, add an amount of supersound process of ethanol (power 250W, frequency 33KHz) 5min, take out, put to room temperature, it is fixed to scale to add ethanol, 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-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.

Radix Notoginseng, ginsenoside Rg in the little hydro-acupuncture preparation of experimental example 15 prescriptions of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more thin layer chromatography discrimination method

Get the little hydro-acupuncture preparation 30ml of prescription of the present invention, extract with n-butyl alcohol 10ml, filter, filtrate is as need testing solution; Other gets Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more, preparation control medicinal material solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, and the reflux, extract, that adds methylene chloride discards dichloromethane solution, residue adds ethyl acetate extraction, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolves with methanol 2ml, in contrast medical material solution; 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 second 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 dichloromethane-Jia acetoacetic ester-methanol-water 22: 45: 10: 15 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless.

Ginsenoside Rg in the little hydro-acupuncture preparation of experimental example 16 prescriptions of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more liquid chromatograph differentiate

Get the little hydro-acupuncture preparation 25ml of prescription of the present invention, put in the 50ml measuring bottle, add ethanol, take out, put, add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m) to scale to room temperature to scale; With the ginsenoside Rg ₁, ginsenoside Re, Panax Notoginseng saponin R ₁In one or more reference substances alcoholic solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-1% glacial acetic acid is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

The assay of the little hydro-acupuncture preparation scutellarin of experimental example 17 prescriptions of the present invention

Get the little hydro-acupuncture preparation of prescription of the present invention as need testing solution; Methanol solution with the scutellarin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-1% glacial acetic acid aqueous solution is a mobile phase at 55%: 45%, 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.

Content of total flavone is measured in the little hydro-acupuncture preparation of experimental example 18 prescriptions of the present invention

Originally get the little hydro-acupuncture preparation of prescription of the present invention as need testing solution; With the methanol solution of scutellarin reference substance product solution in contrast.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.

Ginsenoside Rg in the little hydro-acupuncture preparation of experimental example 19 prescriptions of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more assay

Get the little hydro-acupuncture preparation 25ml of prescription of the present invention, put in the 50ml measuring bottle, add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m) to scale; 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-1% glacial acetic acid 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:

The assay of total saponins in the little hydro-acupuncture preparation of experimental example 20 prescriptions of the present invention

Get the little hydro-acupuncture preparation 50ml of prescription of the present invention, evaporate to dryness, residue add methanol makes dissolving and quantitative the transfer put in the 25ml measuring bottle in right amount, adds methanol to scale, shake up, precision is measured 2.5ml, puts in the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 1.25ml, perchloric acid 5ml 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.

One or more thin layer chromatography discrimination method in Herba Erigerontis medical material, the scutellarin in the experimental example 21 prescription capsule preparations of the present invention

Get prescription capsule preparations 2g of the present invention, add methanol 10ml and extract, centrifugal, get 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 formate and extract, 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 6: 4: 2, 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.

The liquid chromatograph discrimination method of experimental example 22 prescription capsule preparations scutellarins of the present invention

Get prescription capsule preparations 0.25g of the present invention, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of ethanol (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 the scutellarin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-1% aqueous formic acid is a mobile phase at 48%: 52%, 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.

Radix Notoginseng, ginsenoside Rg in the experimental example 23 prescription capsule preparations of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more thin layer chromatography discrimination method

Get prescription capsule preparations 3g of the present invention, extract with ethyl acetate 10ml, filter, filtrate is as need testing solution; Other gets Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more, preparation control medicinal material solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adds the chloroform reflux, extract,, discards chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolves with methanol 2ml, in contrast medical material solution; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more reference substances, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with dichloromethane-ethyl acetate-alcohol-water 14: 30: 10: 15 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless.

Ginsenoside Rg in the experimental example 24 prescription capsule preparations of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more liquid chromatograph differentiate

Get prescription capsule preparations 2.5g of the present invention, 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 Re, Panax Notoginseng saponin R ₁In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.2% phosphoric acid is mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 30%, and from 15 minutes to 30 minutes, the ratio of acetonitrile rose to 40% by 30%, from 30 minutes to 50 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

The assay of experimental example 25 prescription capsule preparations scutellarins of the present invention

Get prescription capsule preparations 0.25g of the present invention, 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 acetonitrile-0.4% phosphate aqueous solution is a mobile phase at 46%: 54%, 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.

Content of total flavone is measured in the experimental example 26 prescription capsule preparations of the present invention

Prescription capsule preparations 0.25g of the present invention puts in the 25ml measuring bottle, adds an amount of supersound process of 80% alcoholic solution (power 250W, frequency 33KHz) and makes dissolving, takes out, and puts to room temperature, adds 80% alcoholic solution 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.

Ginsenoside Rg in the experimental example 27 prescription capsule preparations of the present invention ₁, ginsenoside Rb ₁, Panax Notoginseng saponin R ₁In one or more assay

Get prescription capsule preparations 2.5g of the present invention, put in the 50ml measuring bottle, add ethanol 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, take out, put, add ethanol 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 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 methanol-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of methanol is 25%, and from 15 minutes to 30 minutes, the ratio of methanol rose to 38% by 25%, from 30 minutes to 35 minutes, the ratio of methanol 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:

Claims

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

2, according to the method for quality control of the described compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng of claim 1, it is characterized in that: this method adopts liquid chromatography test based on the finger printing of Herba Erigerontis composition characteristics with based in the finger printing of Radix Notoginseng composition characteristics one or both:

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%.

3, according to the method for quality control of the described compound Chinese medicinal preparation made from Herba Erigerontis, Radix Notoginseng of claim 2, it is characterized in that: this method adopts liquid chromatography test based on the finger printing of Herba Erigerontis composition characteristics with based in the finger printing of Radix Notoginseng composition characteristics one or both:

(1) preparation of need testing solution: it is an amount of to get compound Chinese medicinal preparation to be measured, and thin up shakes up to suitable concn, filters, and gets subsequent filtrate as need testing solution;

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

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

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

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

The assay of total saponins in d, 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 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:

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, it is characterized in that: the assay result of described compound Chinese medicinal preparation, calculate scutellarin, total flavones, total saponins, Panax Notoginseng saponin R according to percentage by weight ₁, the ginsenoside Rg ₁With ginsenoside Rb ₁In the total content of all or part of material account for deduction adjuvant and outer more than 25% of total solid of moisture in the preparation.