CN1939434A

CN1939434A - Quality control of medicine composition

Info

Publication number: CN1939434A
Application number: CNA2005101078216A
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 ginseng (or red ginseng or pilose asiabell root) or its extract and the paeoniforin includes the fingerprint test method and/or differentiation test method and/or content measuring method.

Description

A kind of method of quality control of pharmaceutical composition

Technical field

The present invention is a kind of method of quality control of pharmaceutical composition, 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 pharmaceutical composition method for quality; At present, in the related drugs preparation, generally only with the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, peoniflorin be for detecting index, but its quality of reactor product comprehensively at all is not very reasonable with this quality that is used for controlling pharmaceutical composition 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 pharmaceutical composition.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 pharmaceutical composition are formed and proportioning following (by weight):

Scheme one:

Radix Ginseng (or Radix Ginseng Rubra or Radix Codonopsis) 1～99 weight portion peoniflorin 30～0.1 weight portions

Scheme two:

Radix Ginseng (or Radix Ginseng Rubra or Radix Codonopsis) extract 0.1～20 weight portion peoniflorin 30～0.1 weight portions

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

The present invention constitutes like this:

The method of quality control of the pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin, it is characterized in that: this method comprises following all or part of content:

(1) finger printing of pharmaceutical composition test comprises with Radix Ginseng Rubra or constituent of ginseng being characterized as main finger printing, a kind of based in the finger printing of Radix Codonopsis composition characteristics;

(2) Radix Ginseng Rubra or ginseng crude drug, ginsenoside Rg in the pharmaceutical composition ₁, ginsenoside Rb ₁, among the ginsenoside Re, ginsenoside Rf, peoniflorin, the differential test method of lobetyolin, all or part of composition of atractylenoide;

(3) ginsenoside Rg in the pharmaceutical composition ₁, ginsenoside Rb ₁, ginsenoside Re, peoniflorin, total saponins, lobetyolin, all or part of composition of atractylenoide content test method.

The method of quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin is characterized in that: this method adopt liquid chromatography test with Radix Ginseng Rubra or constituent of ginseng be characterized as main finger printing, based on the finger printing of Radix Codonopsis composition characteristics:

A, employing liquid chromatography test Radix Ginseng Rubra or constituent of ginseng are characterized as main finger printing:

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

(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Ginseng Rubra or Radix Ginseng and the Radix Ophiopogonis medical material, comprise the ginsenoside Rg ₁, ginsenoside Rb ₁, a kind of among the ginsenoside Re, ginsenoside Rf, 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 A is 0.01mol/L～2mol/L sodium dihydrogen phosphate or 0.01mol/L～2mol/L potassium dihydrogen phosphate or water or 0.1%～5% glacial acetic acid solution or 0.1%～5% formic acid solution or 0.02%～5% phosphoric acid solution; B is 10%～90% acetonitrile solution or 10%～90% methanol solution, and gradient elution, flow velocity be 0.5～2.0ml/min, detect wavelength is that one or several or evaporation photodetector in 190～410nm scope detects, and column temperature is in 20～60 ℃ of scopes;

(4) formulation of standard finger-print: the means of testing that is characterized as main standard finger-print with said method as formulation with Radix Ginseng Rubra or constituent of ginseng; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, in the described standard finger-print, total peak has 5～30, retention time with definite object of reference chromatographic peak is a benchmark, calculate the relative retention time of other total chromatographic peak, its relative standard deviation must not surpass ± 20%;

(5) be characterized as the means of testing of main finger printing with the described method in (1)～(3) as Radix Ginseng Rubra or constituent of ginseng in the pharmaceutical composition to be measured, the finger printing of preparation testing sample;

(6) with the finger printing of pharmaceutical composition 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 pharmaceutical composition to be measured, should be 0.80～1.00;

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

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

B, employing liquid chromatography test Radix Codonopsis composition characteristics are main finger printing:

(1) preparation of need testing solution: it is an amount of to get pharmaceutical composition to be measured, adds water or methanol or dissolve with ethanol or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: get the main active reference substance in an amount of codonopsis pilosula, comprise in lobetyolin, the atractylenoide one or both, water or methanol, dissolve with ethanol are diluted to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 5%～99%: 95%～5% acetonitrile or methanol-water or 0.01mol/L～2mol/L sodium dihydrogen phosphate or 0.2%～3% glacial acetic acid or 0.2%～3% formic acid or 0.2%～3% phosphoric acid solution, flow velocity is 0.5～2.0ml/min, detect wavelength is that one or several or evaporation photodetector in the 190-300nm 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 Codonopsis 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 pharmaceutical composition to be measured based on the means of testing of the finger printing of Radix Codonopsis composition characteristics, the finger printing of preparation testing sample;

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

A, employing liquid chromatography for measuring are characterized as main finger printing with Radix Ginseng Rubra or constituent of ginseng:

(1) preparation of need testing solution: it is an amount of that precision takes by weighing pharmaceutical composition to be measured, adds water and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;

(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 A is the 0.05mol/L potassium dihydrogen phosphate, and Mobile phase B is acetonitrile-water 80: 20, gradient elution, solvent ratios is from 0 minute to 5 minutes, the ratio of Mobile phase B is 25%, and from 5 minutes to 40 minutes, the ratio of Mobile phase B rose to 64% by 25%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 64%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 80% by 64%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 25% by 80%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;

(4) formulation of standard finger-print: the means of testing that is characterized as main standard finger-print with said method as formulation with Radix Ginseng Rubra or constituent of ginseng; Accurate object of reference solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid respectively, according to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, total peak has 5～20, retention time with definite object of reference chromatographic peak is a benchmark, calculates the relative retention time of other total chromatographic peak, and its relative standard deviation must not surpass ± 10%;

(5) with the described method in (1)～(3) as treating that Radix Ginseng Rubra in the pharmaceutical composition or constituent of ginseng are characterized as the means of testing of main finger printing, the finger printing of preparation testing sample;

(6) with the contrast of pharmaceutical composition finger printing to be measured and 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 pharmaceutical composition to be measured, should be 0.90～1.00;

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

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

(1) preparation of need testing solution: it is an amount of that precision takes by weighing pharmaceutical composition to be measured, adds methanol and make the solution that every 1ml contains 50mg, filters, and gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: get the main active atractylenoide in an amount of codonopsis pilosula, add methanol and make the solution that every 1ml contains 0.1mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is methanol-water 67: 33, and flow velocity is that 1.0ml/min, evaporation photodetector detect, 30 ℃ of drift tube temperatures, and carrier gas flux 2.2L/min, column temperature are 30 ℃;

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Radix Codonopsis composition characteristics; Accurate respectively object of reference solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid respectively, 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;

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

The method of quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin, it is characterized in that: the discrimination method of described pharmaceutical composition comprises following all or part of content:

Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in a, the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more thin layer chromatography discrimination method among the ginsenoside Re, ginsenoside Rf:

It is an amount of to get pharmaceutical composition 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 Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, among the ginsenoside Re, ginsenoside Rf one or more, the preparation contrast solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, add the chloroform reflux, extract,, discard chloroform liquid, 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, one or more reference substances among the ginsenoside Re, ginsenoside Rf, add methanol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, lower floor's solution or n-butyl alcohol-ethyl acetate or the Ethyl formate-water 1～10: 0.2～2 placed below 5～40: 10～100: 5～50: 0.2～30 10 ℃ with chloroform-ethyl acetate or Ethyl formate-methanol-water: 1～15 upper strata is developing solvent, launch, take out, dry, spray is with 5～50% sulphuric acid ethanol reagent or 50% sulphate reagent, 80 ℃～160 ℃ dry by the fire to speckle colour developing clear, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with 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 b, the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more liquid chromatograph is differentiated among the ginsenoside Re:

It is an amount of to get pharmaceutical composition 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 ₁The methanol of one or more reference substances among the ginsenoside Re or alcoholic solution 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, 5%～40%: 95%～60% methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

The thin layer chromatography discrimination method of peoniflorin in c, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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 the peoniflorin reference substance, adds methanol or ethanol and makes the solution that every 1ml contains 2mg; 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 ₂₅₄Lamellae, with lower floor's solution of chloroform or dichloromethane-ethyl acetate or Ethyl formate-methanol or ethanol-formic acid or acetic acid 1～120: 0.2～15: 0.5～25: 0.01～5 or chloroform or dichloromethane-ethyl acetate or Ethyl formate-methanol or alcohol-water 3～50: 10～120: 5～60: 1～25 or chloroform or n-butyl alcohol or dichloromethane or toluene-methanol or glacial acetic acid or formic acid-ethyl acetate or water 1～80: 0.1～50: 0.05～25 or chloroform-methanol 1～25: 0.2～15 is developing solvent, launch, take out, dry, spray is with 0.2%～10% ferric chloride alcoholic solution earlier, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the same color speckle; Spray again with behind the vanillin sulfuric acid solution 80 ℃～160 ℃ dry by the fire to speckle colour developing clear or hot blast blow clear or put under ultra-violet lamp 365nm or the 254nm and inspect to the speckle colour developing, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the same color speckle;

The liquid chromatograph discrimination method of peoniflorin in d, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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 peoniflorin 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, 5%～95%: 95%～5% 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 or 0.005mol/L～2mol/L sodium dihydrogen phosphate or 0.005mol/L～2mol/L potassium dihydrogen phosphate is a 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;

The thin layer chromatography discrimination method of lobetyolin in e, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition to be measured, be dissolved in water, with n-butyl alcohol or ethyl acetate or chloroform extraction, extract is concentrated into dried, and residue is with methanol or dissolve with ethanol, as need testing solution, or put in C18 or the C8 solid phase extraction column, use 5%～50% methanol, methanol-eluted fractions successively, collect meoh eluate, be concentrated into 1ml, as need testing solution; Other gets one or both preparation contrast solutions in Radix Codonopsis control medicinal material, the lobetyolin; The preparation of control medicinal material solution: get the Radix Codonopsis control medicinal material, filter after adding water or methanol extraction, filtrate evaporate to dryness, residue with water dissolution after with n-butyl alcohol or ethyl acetate or chloroform extraction, extract is concentrated into dried, residue is with methanol or dissolve with ethanol, and as need testing solution, or filtrate is put in C18 or the C8 solid phase extraction column, use 5%～50% methanol, methanol-eluted fractions successively, collect meoh eluate, be concentrated into 1ml, as need testing solution; The preparation of reference substance solution: get lobetyolin's reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, n-butyl alcohol-glacial acetic acid or formic acid or ethanol or dehydrated alcohol-water 1～35: 0.1～10: 0.05～5 are developing solvent, launch, and take out, dry, spray is with 3%～30% ethanol solution of sulfuric acid, and 80～150 ℃ were heated 1～30 minute, and put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the fluorescence speckle of same color;

The liquid chromatograph discrimination method of lobetyolin in f, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition to be measured, puts in the measuring bottle, adds methanol or ethanol or mobile phase to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with lobetyolin's reference substance is 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, 5%～80%: 95%～20% methanol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～0.5% phosphate aqueous solution are mobile phase, the detection wavelength is one or several in 200～410nm scope, in 20～50 ℃ of scopes of column temperature; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

It is an amount of to get pharmaceutical composition 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, among the ginsenoside Re, ginsenoside Rf one or more, preparation control medicinal material solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, add the chloroform reflux, extract,, discard 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, one or more reference substances among the ginsenoside Re, ginsenoside Rf, 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 pharmaceutical composition 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, one or more reference substances among the ginsenoside Re methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, centrifugal, gets supernatant as need testing solution; Other gets the peoniflorin reference substance, adds methanol respectively and makes the solution that every 1ml contains 2mg; 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-formic acid 6: 1.5: 1.5: 0.5 was developing solvent, launches, take out, dry, spray is with 5% ferric chloride alcoholic solution, in the test sample chromatograph earlier, with the corresponding position of reference substance chromatograph on, show the same color speckle; Spray again with behind the vanillin sulfuric acid solution 105 ℃ dry by the fire to speckle colour developing clear or, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle;

It is an amount of to get pharmaceutical composition 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 solution with the peoniflorin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% formic acid solution was a mobile phase in 40%: 60%, and the detection wavelength is 230nm; 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 pharmaceutical composition to be measured, add methanol 25ml, supersound process 30 minutes filters, the filtrate evaporate to dryness, residue adds water 2ml makes dissolving, puts in the C18 solid phase extraction column (500mg is with methanol, each the 10ml prewashing of 20% methanol), use 20% methanol, each 5ml eluting of methanol successively, collect meoh eluate, be concentrated into 1ml, as need testing solution; Other gets the Radix Codonopsis control medicinal material, shines medical material solution in pairs with legal system; It is an amount of to get lobetyolin's reference substance again, adds methanol and makes every 1ml and contain 1mg solution, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw need testing solution 6 μ l, reference substance solution 2 μ l, put respectively on same high-efficient silica gel G lamellae, with n-butyl alcohol-glacial acetic acid-water is developing solvent at 7: 1: 0.5, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ were heated 5 minutes, and put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

It is an amount of to get pharmaceutical composition 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; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

The method of quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin, it is characterized in that: the method for testing of described pharmaceutical composition content should comprise following all or part of content:

Ginsenoside Rg in a, the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more assay among the ginsenoside Re:

It is an amount of to get pharmaceutical composition 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 ₁The methanol of one or more reference substances among the ginsenoside Re or alcoholic solution 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, 5%～40%: 95%～60% methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, pharmaceutical composition 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 0.8mg;

(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.4mg;

(3) the per unit amount contains ginsenoside Rb ₁Limit must not be less than 0.5mg;

(4) the per unit amount contains the ginsenoside Rg ₁, the ginsenoside Re the limit of summation must not be less than 1.7mg;

The assay of total saponins in b, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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 ginsenoside Re or ginsenoside Rf be reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures trap, calculate with external standard method or standard curve method, pharmaceutical composition 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 ginsenoside Re or ginsenoside Rf's meter, must not be less than 3mg;

Content of paeoniflorin is measured in c, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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 peoniflorin 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, 5%～95%: 95%～5% 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 or 0.005mol/L～2mol/L sodium dihydrogen phosphate or 0.005mol/L～2mol/L potassium dihydrogen phosphate is a 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 peoniflorin must not be less than 10mg;

The assay of lobetyolin in d, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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; Methanol solution with lobetyolin's reference substance is 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, 5%～80%: 95%～20% methanol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～0.5% phosphate aqueous solution are mobile phase, the detection wavelength is one or several in 200～4l0nm scope, in 20～50 ℃ of scopes of column temperature; 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 lobetyolin must not be less than 0.05mg.

The method of quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin, it is characterized in that: the method for testing of described pharmaceutical composition content is following one or more methods:

It is an amount of to get pharmaceutical composition 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, one or more reference substances among the ginsenoside Re methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with external standard method or standard curve method, pharmaceutical composition 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 1.6mg;

(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.8mg;

(3) the per unit amount contains ginsenoside Rb ₁Limit must not be less than 1.0mg;

(4) the per unit amount contains the ginsenoside Rg ₁, the ginsenoside Re the limit of summation must not be less than 3.4mg;

The assay of total saponins in b, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition to be measured, puts in the 10ml measuring bottle, adds water to scale, shake up, precision is measured 0.6ml, to 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 cooling 2 minutes, precision added glacial acetic acid to 25ml 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 external standard method or standard curve method at the wavelength place of 547nm, pharmaceutical composition 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 6mg;

Content of paeoniflorin is measured in c, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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 solution with the peoniflorin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, methanol-0.1% formic acid solution was a mobile phase in 40%: 60%, detecting wavelength is that 230nm calculates with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains peoniflorin must not be less than 20mg;

The assay of lobetyolin in d, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; 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 lobetyolin must not be less than 0.1mg.

The method of quality control of described pharmaceutical composition is characterized in that: the assay result of the ejection preparation of described pharmaceutical composition, can survey composition except that adjuvant, the ginsenoside Rg in its quality standard ₁, ginsenoside Rb ₁, all or part of kind among ginsenoside Re, peoniflorin, total saponins, lobetyolin, atractylenoide etc. total content account for more than 25%.

Compared with prior art, the present invention's quality of the pharmaceutical composition made with Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin of perfect control more.The Chinese medicine ingredients complexity, if only with wherein one, two kind of composition illustrate its inherent quality, has certain one-sidedness, more can't judge the index components of its drug effect.Therefore the applicant has formulated with Radix Ginseng Rubra or constituent of ginseng and has been characterized as the quality that main finger printing, finger printing, discriminating and assay based on the Radix Codonopsis composition characteristics are controlled pharmaceutical composition comprehensively.But because contained complex chemical composition between each medical material in the pharmaceutical composition, 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 Radix Ginseng in the pharmaceutical composition or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin 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 pharmaceutical composition product made with Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin, and method precision, stability are all higher.

Experimental example 1 is characterized as the preparation of main finger printing with Radix Ginseng Rubra or constituent of ginseng

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 (15: 85), (3) acetonitrile-water (gradient elution) (4) A is 50mmol.L ^-1KH ₂PO ₄Solution, B is that (the gradient elution volume proportion is from 0 minute to 5 minutes to acetonitrile-water (80: 20), the ratio of Mobile phase B is 25%, and from 5 minutes to 40 minutes, the ratio of Mobile phase B rose to 64% by 25%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 64%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 80% by 64%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 25% by 80%) 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:

Be 50mmol.L at A in the research ^-1KH ₂PO ₄Solution, B are under acetonitrile-water (80: 20) (gradient elution) the mobile phase condition, have investigated the chromatographic peak situation under different-waveband typical wavelengths 203,210,230,254 respectively, the result shows, chromatographic peak is more under 203nm, and peak shape is better, so finally select for use 203nm 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 Ginseng Rubra or Radix Ginseng 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:

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.

The thin layer chromatography discrimination method of peoniflorin in experimental example 2 pharmaceutical compositions:

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

Condition	Problem
Condition	Problem	Chloroform-ethyl acetate-methanol (7-6-5) silica gel g thin-layer plate methylene chloride-methanol-formic acid (7-6-5) silica gel G F ₂₅₄Lamellae chloroform-ethanol-glacial acetic acid, (7-2-2) silica gel H lamellae chloroform-ethyl acetate-formic acid, (7-1-2) silica gel g thin-layer plate chloroform-ethyl acetate-glacial acetic acid, (7-1-2) silica gel H lamellae chloroform-ethyl acetate-methanol-formic acid, (8-3-3-2) silica gel G F ₂₅₄Lamellae chloroform-ethyl acetate-methanol-formic acid (6-1.5-1.5-0.5) silica gel g thin-layer plate	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, 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 silica gel g thin-layer plate to be immobile phase, be developing solvent with chloroform-ethyl acetate-methanol-formic acid (6-1.5-1.5-0.5), with this understanding, the Rf value of Radix Paeoniae is moderate, and it is clear to separate with other speckle, negative noiseless.

The liquid chromatograph discrimination method of peoniflorin in experimental example 3 pharmaceutical compositions:

Feature for the outstanding Radix Paeoniae Rubra or the Radix Paeoniae Alba, except the thin layer discrimination method, selected peoniflorin as its characteristic component, but owing to there is composition like more, the polar phase close in the medical material with the peoniflorin structure, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase peoniflorin 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 (40: 60) 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 (40: 60) is a mobile phase, and with this understanding, the peoniflorin retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless.

Experimental example 4 pharmaceutical composition ginsenoside Rgs ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method

For the feature of outstanding Radix Ginseng or Radix Ginseng Rubra, selected the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf be as its feature speckle, but owing to exist more and the ginsenoside Rg in the medical material ₁, ginsenoside Rb ₁, composition like close, the polar phase of ginsenoside Re, ginsenoside Rf's structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:

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

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

Ginsenoside Rg in experimental example 5 pharmaceutical compositions ₁, ginsenoside Rb ₁, the ginsenoside Re the liquid chromatograph discrimination method

For the feature of outstanding Radix Ginseng or Radix Ginseng Rubra, selected the ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re is as 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, the ginsenoside Re separates:

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 35 minutes, the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, from 55 minutes to 70 minutes, the ratio of acetonitrile is 29 %, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% octadecylsilane chemically bonded silica by 29%	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 are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rises to 40% by 29%, with this understanding, and the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re's retention time is moderate, the peak is capable sharp-pointed, symmetry is negative noiseless.

Experimental example 6 total saponin contents are measured

1 instrument, reagent

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

SARTORIUS BP211D electronic analytical balance

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

Methanol chromatographically pure J.T.Baker

Glacial acetic acid analytical pure Shanghai reagent one factory

Chemical plant, prosperous source, perchloric acid analytical pure Tianjin

The pure water WAHAHA

2 methods and result

Take by weighing the ginsenoside Rg 2.1 detect the selection precision of wavelength ₁5.14mg, put in the 100ml measuring bottle, add an amount of methanol supersound process (power 250W, frequency 33KHz) makes dissolving, add methanol, shake up to scale, 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 pharmaceutical composition.

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

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

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

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

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

The ginsenoside Rg ₁The standard curve determination data

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

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

The precision experiment

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

5 stability tests

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

The reference substance solution stability experiment

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

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

The need testing solution stability experiment

Time (min)	0	10	20	40	60	Average	RSD％
Time (min)	0	10	20	40	60	Average	RSD％	Content (mg/ bottle)	12.84	13.11	12.96	13.16	12.79	12.97	1.25

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

Repeated experiment

Numbering	1	2	3	4	5	X is average	RSD％
Numbering	1	2	3	4	5	X is average	RSD％	Content (mg/ bottle)	13.12	13.25	12.94	12.88	13.06	13.05	1.12

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 (0.634mg/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: 25.31mg/g in the pharmaceutical composition

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	26.51 25.35 24.84 26.33 25.79	0.6710 0.6416 0.6287 0.6664 0.6527	0.634 0.634 0.634 0.634 0.634	0.6710 0.6416 0.6287 0.6664 0.6527	97.74 98.36 99.05 97.12 98.36

Average recovery rate=98.13% RSD=0.74%

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	16.12 13.54 14.97

Experimental example 7 ginsenoside Rgs ₁, ginsenoside Rb ₁, the ginsenoside Re content

1. instrument and reagent

(1) instrument: Agilent1100 high performance liquid chromatograph, chemstationsys work station.The TU-1800SPC ultraviolet spectrophotometer.

(2) reagent: ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;

2. detect the selection of wavelength: precision takes by weighing the ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re, split in the 10ml measuring bottle, add methanol dilution and make the solution that every 1ml contains 0.5mg, scan in the 200-400nm wave-length coverage.The ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re all has absorption maximum at the 203nm place, therefore selects the detection wavelength of 203nm as assay.

3. chromatographic condition: Dikma ODS (4.6mm * 250mm, 5um); Mobile phase: acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 35 minutes, the ratio of acetonitrile is 19%, and from 35 minutes to 55 minutes, the ratio of acetonitrile rose to 29% by 19%, from 55 minutes to 70 minutes, the ratio of acetonitrile is 29%, and from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; The detection wavelength is 203nm; Flow velocity: 1.0ml/min.

4. the preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁An amount of with the ginsenoside Re, accurate claim surely, add methanol and make 1ml and contain the ginsenoside Rg ₁0.20mg, ginsenoside Rb ₁0.20mg, the mixed solution of ginsenoside Re 0.1mg.

5. the preparation of need testing solution: get this product under the content uniformity item, get content, mixing is therefrom got about 0.5g, and accurate the title decides, and puts in the 5ml measuring bottle, adds to flow mutual-assistance dissolving and be diluted to scale, shakes up, promptly.

With this understanding, negative sample ginsenoside Rg in the disturbed specimen not ₁, ginsenoside Rb ₁With ginsenoside Re's mensuration, and separating degree is good.

6. ginsenoside Rg ₁, ginsenoside Rb ₁Take by weighing the ginsenoside Rg with ginsenoside Re's linear relationship precision ₁10.24mg, ginsenoside Rb ₁10.09mg, ginsenoside Re 7.54mg, put altogether in the 10ml measuring bottle, it is fixed to scale to add mobile phase, shakes up, product solution (contains the ginsenoside Rg among every 1ml in contrast ₁1.024mg, ginsenoside Rb ₁1.009mg, contain ginsenoside Re 0.754mg), precision is measured 2.5ml, puts in the 10ml measuring bottle, adds mobile phase to scale, shakes up, in contrast the product dilute solution.(contain the ginsenoside Rg among every 1ml ₁0.256mg, contain ginsenoside Rb ₁0.25225mg, contain ginsenoside Re 0.1885mg) and accurate reference substance solution 3 μ l, 6 μ l, the 10 μ l of drawing; Reference substance dilute solution 2 μ l, 5 μ l, 10 μ l; Injecting chromatograph of liquid, is vertical coordinate with the peak area, the ginsenoside Rg ₁Be figure for abscissa, the drawing standard curve with ginsenoside Re's amount.

The ginsenoside Rg ₁Linear relationship

Numbering	The ginsenoside Rg ₁(μg)	Peak area
Numbering	The ginsenoside Rg ₁(μg)	Peak area	1 2 3 4 5 6	0.512 1.280 2.560 3.072 6.144 10.24	150.63 475.54 890.23 1058.85 2107.02 3538.96

Regression equation: Y=345.01X+1.20;

The coefficient of determination: γ=0.9996;

The ginsenoside Rg ₁Good in 0.512～10.240 μ g scope internal linear relation.

Ginsenoside Rb ₁Linear relationship

Numbering	Ginsenoside Rb ₁(μg)	Peak area
Numbering	Ginsenoside Rb ₁(μg)	Peak area	1 2 3 4 5 6	0.5045 1.2613 2.5225 3.0270 6.0540 10.090	142.08 353.07 715.48 860.21 1709.14 2855.26

Regression equation: Y=282.95X-0.44;

The coefficient of determination: γ=0.9999;

Ginsenoside Rb ₁Good in 0.5045～10.090 μ g scope internal linear relation.

Ginsenoside Re's linear relationship

Numbering	Ginsenoside Re (μ g)	Peak area
Numbering	Ginsenoside Re (μ g)	Peak area	1 2 3 4 5 6	0.3770 0.9425 1.8850 2.2620 4.5240 7.5400	184.37 466.91 928.26 1104.76 2216.15 3698.24

Regression equation: Y=490.12X+1.11;

The coefficient of determination: γ=0.9999;

The ginsenoside Re is good in 0.3770～7.5400 μ g scope internal linear relation.

7. reference substance precision test precision is drawn ginsenoside Rg under the standard curve item ₁, the ginsenoside Rg ₁(contain the ginsenoside Rg among every 1ml with ginsenoside Re's reference substance dilute solution ₁0.256mg, ginseng saponin Rb ₁0.25225mg, contain ginsenoside Re 0.1885mg) and 10 μ l, inject chromatograph of liquid, the record peak area, continuous sample introduction is measured 5 times, investigates the precision of reference substance solution.

The test of reference substance solution precision

Test number (TN)	1	2	3	4	5	Average	RSD(％)
Test number (TN)	1	2	3	4	5	Average	RSD(％)	The ginsenoside Rg ₁Ginsenoside Rb ₁The ginsenoside Re	884.26 726.23 921.63	893.71 722.57 922.44	899.04 724.92 925.36	892.35 725.86 917.03	895.89 722.78 930.58	893.05 724.47 923.41	0.62 0.24 0.54

The result shows, the ginsenoside Rg ₁, ginsenoside Rb ₁Good with ginsenoside Re's reference substance solution precision.

8. stability test

8.1 ginsenoside Rg under the accurate absorption of the reference substance stability test standard curve item ₁, the ginsenoside Rg ₁(contain the ginsenoside Rg among every 1ml with ginsenoside Re's reference substance dilute solution ₁0.256mg, ginseng saponin Rb ₁0.25225mg, contain ginsenoside Re 0.1885mg) and 10 μ l, inject chromatograph of liquid, the record peak area is measured at 0,6,12,24,48 hour sample introduction.

Reference substance solution stability test result

Testing time (h)	0	6	12	24	48	On average	RSD(％)
Testing time (h)	0	6	12	24	48	On average	RSD(％)	The ginsenoside Rg ₁Ginsenoside Rb ₁The ginsenoside Re	884.26 726.23 921.63	893.71 722.57 922.44	899.04 724.92 925.36	892.35 725.86 917.03	895.89 722.78 930.58	893.05 724.47 923.41	0.62 0.24 0.54

The result shows, the ginsenoside Rg ₁, ginsenoside Rb ₁Have good stability with ginsenoside Re's reference substance solution.

8.2 the need testing solution stability test is got this product under the weight differential item, get content, mixing is therefrom got about 0.5g, and accurate the title decides, put in the 5ml measuring bottle, add and flow mutual-assistance dissolving and decide to shake up the accurate 10 μ l that draw to scale, inject chromatograph of liquid, measure at 0,6,12,24,48 hour sample introduction respectively.

Need testing solution stability test result

Testing time (h)	0	6	12	24	48	Average	RSD(％)
Testing time (h)	0	6	12	24	48	Average	RSD(％)	The ginsenoside Rg ₁(mg/ bottle) ginsenoside Rb ₁(mg/ bottle) ginsenoside Re (mg/ bottle)	0.1084 0.1125 0.5307	0.1107 0.1139 0.5259	0.1094 0.1115 0.5158	0.1112 0.1161 0.5226	0.1086 0.1143 0.5237	0.1097 0.1137 0.5237	1.14 1.55 1.03

The result shows that need testing solution has good stability.

9. replica test

Get under this product content uniformity item 5 bottles on powder pin, get content, porphyrize is therefrom got about 0.5g, accurately claims surely, puts in the 5ml measuring bottle, adds the mutual-assistance dissolving and shake up to scale surely of flowing, and the accurate 10 μ l that draw inject chromatograph of liquid, measure, promptly.

Replica test

Test number	1	2	3	4	5	Average	RSD(％)
Test number	1	2	3	4	5	Average	RSD(％)	The ginsenoside Rg ₁(mg/ bottle) ginsenoside Rb ₁(mg/ bottle) ginsenoside Re (mg/ bottle)	1.042 1.125 0.5258	1.028 1.144 0.5197	1.047 1.128 0.5236	1.031 1.107 0.5104	1.064 1.122 0.5249	1.042 1.125 0.5209	1.38 1.18 1.21

10. average recovery test

Adopt the application of sample absorption method, get this product under the weight differential item, get content, mixing is therefrom got about 0.25g (totally 5 parts), and accurate the title decides, and splits in the 5ml measuring bottle; Precision takes by weighing the ginsenoside Rg ₁13.71mg, ginsenoside Rb ₁13.96mg ginsenoside Re 6.73mg puts in the 25ml measuring bottle altogether, add an amount of supersound process of mobile phase and make dissolving, take out, put to room temperature, add mobile phase to scale, shake up, precision is measured 1ml (totally 5 parts), split in the above-mentioned 5ml measuring bottle, add mobile phase, shake up to scale, the accurate 10 μ l that draw, inject chromatograph of liquid, measure, promptly.

Ginsenoside Rg in the pharmaceutical composition ₁Content: 2.021mg/g;

Ginsenoside Rb in the pharmaceutical composition ₁Content: 2.182mg/g;

Ginsenoside Re's content: 1.010mg/g in the pharmaceutical composition;

The ginsenoside Rg ₁The average recovery test

Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	0.25042 0.25387 0.25124 0.25307 0.25282	0.5061 0.5131 0.5078 0.5115 0.5109	0.5484 0.5484 0.5484 0.5484 0.5484	0.5061 0.5131 0.5078 0.5115 0.5109	98.52 99.36 97.84 98.26 99.05

The ginsenoside Rg ₁Average recovery rate=98.61%; RSD=0.62%

Ginsenoside Rb ₁The average recovery test

Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	0.25042 0.25387 0.25124 0.25307 0.25282	0.5464 0.5539 0.5482 0.5522 0.5517	0.5584 0.5584 0.5584 0.5584 0.5584	1.0898 1.1014 1.0983 1.1067 1.0959	97.31 98.04 98.52 99.31 97.47

Ginsenoside Rb ₁Average recovery rate=98.13%; RSD=0.83%

The test of ginsenoside Re's average recovery

Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1 2 3 4 5	0.25042 0.25387 0.25124 0.25307 0.25282	0.2529 0.2564 0.2538 0.2556 0.2553	0.2692 0.2692 0.2692 0.2692 0.2692	0.5171 0.5230 0.5179 0.5177 0.5196	98.15 99.03 98.14 97.36 98.15

Ginsenoside Re's average recovery rate=98.17%; RSD=0.60%

11. three batches of pilot scale sample sizes are measured

Three batch sample assay results

Lot number	The ginsenoside Rg ₁(mg/ bottle)	Ginsenoside Rb ₁(mg/ bottle)	Ginsenoside Re's (mg/ bottle)
Lot number	The ginsenoside Rg ₁(mg/ bottle)	Ginsenoside Rb ₁(mg/ bottle)	Ginsenoside Re's (mg/ bottle)	1 2 3	1.084 1.007 1.029	1.153 1.114 1.128	0.5152 0.5208 0.5239

Experimental example 8 paeoniflorin contents are measured

1 instrument, reagent

Instrument: JASC0 1580 type high performance liquid chromatographs

Diamond C18 chromatographic column 250 * 4.6mm 5um Di Ma company

SARTARIUS BP211D electronic analytical balance

Reagent: acetonitrile chromatographically pure Fisher company

Methanol analytical pure Beijing Chemical Plant

Sodium dihydrogen phosphate analytical pure Beijing chemical reagents corporation

2 methods and result

2.1 chromatographiccondition

Immobile phase: octadecylsilane chemically bonded silica 250 * 4.6mm 5um

Mobile phase: methanol-1% formic acid solution (40: 60)

Flow velocity: 1ml/min

Column temperature: room temperature

Sample size: 10ul

Detect wavelength: 230nm

2.2 the above-mentioned chromatographic condition of system suitability experimental evidence obtains peoniflorin reference substance, sample chromatogram figure, number of theoretical plate n is all greater than 3000.

2.3 the selection of measuring wavelength is through UV scanning, peoniflorin has absorption maximum at 230nm wavelength place, so selected 230nm is for measuring wavelength.

2.4 the preparation precision of reference substance solution takes by weighing through dry 36 hours peoniflorin reference substance 10mg of phosphorus pentoxide vacuum drying apparatus, puts in the 100ml measuring bottle, adds methanol and makes dissolving in right amount and be diluted to scale, shakes up, and promptly gets (containing peoniflorin 0.1mg among every 1ml).

2.5 this product under the content uniformity item is got in the preparation of need testing solution, gets content, mixing, get about 0.1g, the accurate title, decide, and puts in the 100ml measuring bottle, it is an amount of to add methanol, and supersound process (power 250W, frequency 33KHz) makes dissolving, take out, put coldly, be diluted to scale with methanol, shake up, filter, filter with microporous filter membrane (0.45um), get subsequent filtrate, promptly.

2.6 negative control test for investigating the mensuration whether other adjuvant disturbs peoniflorin, except that the Radix Paeoniae Rubra or the Radix Paeoniae Alba, takes by weighing other adjuvant in the prescription ratio and makes negative control solution and mensuration with need testing solution with method.The result shows that negative sample is measured noiseless to content of paeoniflorin.

The investigation precision of 3 linear relationships takes by weighing through dry 36 hours peoniflorin reference substance 11.48mg of phosphorus pentoxide vacuum drying apparatus, put in the 10ml measuring bottle, adding methanol makes dissolving in right amount and is settled to scale, shake up, precision measures 0.4,0.8,1.2,1.6,2.0ml, puts respectively in the 10ml measuring bottle, be diluted to scale with methanol, shake up, the therefrom accurate respectively 10ul of absorption injects high performance liquid chromatograph, measures by above-mentioned chromatographic condition.With the peak area is vertical coordinate, and peoniflorin amount (ug) is abscissa mapping, drawing standard curve.

Regression equation is Y=1270296.82X+2219.70

Linear coefficient is γ=0.9999

Peoniflorin is good in 0.4592～2.2960ug scope internal linear.

The standard curve determination data

Numbering	Peoniflorin amount (ug)	Peak area
Numbering	Peoniflorin amount (ug)	Peak area	1 2 3 4 5	0.4592 0.9184 1.3776 1.8368 2.2960	565516.5 1171490 1783885 2344300 2895713

4 stability tests are got the content 105.19mg under this product content uniformity item, press the operation down of the preparation of text need testing solution and algoscopy item, and every 1 hour replication 1 time, the result shows that within 4 hours need testing solution is stable.

Time (h)	0	1	2	3	4	On average	RSD
Time (h)	0	1	2	3	4	On average	RSD	Peoniflorin (mg/ bottle)	47.91	48.03	48.11	48.64	48.53	48.44	1.00

The test of 5 precision is accurate to take by weighing through dry 36 hours peoniflorin reference substance 11.56mg of phosphorus pentoxide desiccator, presses the preparation and the operation down of algoscopy item of text reference substance solution, repeats to survey 5 times, and the result is as follows:

Test number	1	2	3	4	5	On average	RSD
Test number	1	2	3	4	5	On average	RSD	Peak area	1374344	1376226	1378443	1375983	1376550	1376309	0.11

6 replica tests are got 5 parts of this product, and accurate the title decides, and press the preparation and the operation down of algoscopy item of text need testing solution, and the result is as follows:

Test number	1	2	3	4	5	On average	RSD
Test number	1	2	3	4	5	On average	RSD	Peoniflorin (mg/ bottle)	48.26	48.04	49.32	48.57	47.83	48.40	1.20

7 recovery tests adopt the application of sample absorption method, get this product under the content uniformity item, get content, and mixing is therefrom got about 50mg (totally 5 parts), and accurate the title decides, and splits in the 100ml measuring bottle; Precision is measured peoniflorin reference substance solution (1.6644mg/ml) 3ml (totally 5 parts), split in the above-mentioned 100ml measuring bottle, add an amount of supersound process of methanol and make dissolving, take out, put cold, add methanol to scale, shake up, filter with microporous filter membrane (0.45um), the accurate subsequent filtrate 10 μ l that draw inject chromatograph of liquid, measure, promptly.

Paeoniflorin content is 93.86mg/g in the pharmaceutical composition

Numbering	Content weighing (mg)	Peoniflorin amount (mg) in the content	The about glycosides addition of Chinese herbaceous peony (mg)	Measured value (mg)	The response rate (%)
Numbering	Content weighing (mg)	Peoniflorin amount (mg) in the content		Measured value (mg)	The response rate (%)	1 2 3 4 5	53.21 51.86 52.94 53.23 52.28	4.994 4.868 4.969 4.996 4.907	4.9932 4.9932 4.9932 4.9932 4.9932	9.8002 9.7125 9.8853 9.7687 9.7958	96.25 97.03 98.46 95.58 97.91

Average recovery rate=97.05%, RSD=1.21%.

The mensuration of 8 samples is pressed the preparation and the operation down of algoscopy item of text need testing solution, measures three batch samples, and the result is as follows:

Lot number	Paeoniflorin content (mg/ bottle)
Lot number	Paeoniflorin content (mg/ bottle)	1 batch 2 batches 3 batches	48.83 49.21 50.08

The specific embodiment

Embodiment 1: adopt liquid chromatography for measuring to be characterized as main finger printing with Radix Ginseng Rubra or constituent of ginseng

(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, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;

(6) with the contrast of prescription powder injection formulation finger printing of the present invention and 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 relative deviation of the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak must not surpass ± 10%.

Embodiment 2: adopt liquid chromatography test Radix Ginseng Rubra or constituent of ginseng to be characterized as main finger printing:

(1) preparation of need testing solution: it is an amount of to get prescription hydro-acupuncture preparation of the present invention, adds methanol and makes the solution that every 1ml contains 80mg, shakes up, and filters, and gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg ₁, ginsenoside Rb ₁, a kind of among the ginsenoside Re, ginsenoside Rf, add methanol and make the solution that every 1ml contains 0.5mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase A is 0.5% glacial acetic acid solution; B is 50% methanol solution, gradient elution, solvent ratios are from 0 minute to 10 minutes, and the ratio of Mobile phase B is 20%, from 10 minutes to 40 minutes, the ratio of Mobile phase B rises to 65% by 20%, and from 40 minutes to 50 minutes, the ratio of Mobile phase B was 65%, from 50 minutes to 70 minutes, the ratio of Mobile phase B rises to 80% by 65%, and from 70 minutes to 80 minutes, the ratio of Mobile phase B reduced to 20% by 80%; Flow velocity is 0.6ml/min, detection wavelength 201nm, 50 ℃ of column temperatures;

(5) be characterized as the means of testing of main finger printing with said method as Radix Ginseng Rubra or constituent of ginseng in the prescription hydro-acupuncture preparation of the present invention, the finger printing of preparation testing sample;

(6) with the finger printing and the contrast of above-mentioned standard finger-print of prescription hydro-acupuncture preparation of the present invention, meet the following requirements:

I. calculate the similarity of the finger printing and the standard finger-print of prescription hydro-acupuncture preparation of the present invention, should be 0.80～1.00;

II. in the prescription hydro-acupuncture preparation finger printing of the present invention, non-total peak area must not surpass 10% of total peak area;

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

Embodiment 3: Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more thin layer chromatography discrimination method among the ginsenoside Re, ginsenoside Rf

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

Embodiment 4: ginsenoside Rg in the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more liquid chromatograph is differentiated among the ginsenoside Re

It is an amount of to get prescription powder injection formulation of the present invention, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, one or more reference substances among the ginsenoside Re methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0m1/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.

Embodiment 5: the thin layer chromatography discrimination method of peoniflorin in the pharmaceutical composition

It is an amount of to get prescription powder injection formulation of the present invention, adds methanol extraction, centrifugal, gets supernatant as need testing solution; Other gets the peoniflorin reference substance, adds methanol respectively and makes the solution that every 1ml contains 2mg; 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-formic acid 6: 1.5: 1.5: 0.5 was developing solvent, launches, take out, dry, spray is with 5% ferric chloride alcoholic solution, in the test sample chromatograph earlier, with the corresponding position of reference substance chromatograph on, show the same color speckle; Spray again with behind the vanillin sulfuric acid solution 105 ℃ dry by the fire to speckle colour developing clear or, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle.

Embodiment 6: the liquid chromatograph discrimination method of peoniflorin in the pharmaceutical composition

It is an amount of to get prescription powder injection formulation of the present invention, adds dissolve with methanol or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Methanol solution with the peoniflorin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% formic acid solution was a mobile phase in 40%: 60%, and the detection wavelength is 230nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 7: Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more thin layer chromatography discrimination method among the ginsenoside Re, ginsenoside Rf:

It is an amount of to get prescription dispersible tablet of the present invention, uses ethyl acetate 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, among the ginsenoside Re, ginsenoside Rf one or more, the preparation contrast solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, add the chloroform reflux, extract,, discard chloroform liquid, residue adds ethyl acetate extraction, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, one or more reference substances among the ginsenoside Re, ginsenoside Rf, add ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 15 μ l of above-mentioned solution, put respectively on same silica gel H lamellae, with n-butyl alcohol-Ethyl formate-upper strata of 6: 1: 7 of water is developing solvent, launches, and takes out, dry, spray is with 50% sulphate reagent, and 150 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color, negative noiseless.

Embodiment 8: ginsenoside Rg in the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more liquid chromatograph is differentiated among the ginsenoside Re

It is an amount of to get prescription drop pill of the present invention, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, one or more reference substances among the ginsenoside Re alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, methanol-1.5% glacial acetic acid aqueous solution was a mobile phase in 30%: 70%, gradient elution, flow velocity is 1.5ml/min, and the detection wavelength is 201nm, in 50 ℃ of scopes of column temperature; 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.

Embodiment 9: the thin layer chromatography discrimination method of peoniflorin in the pharmaceutical composition:

It is an amount of to get prescription micropill of the present invention, adds ethyl acetate extraction, filters, and gets filtrate as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1ml contains 2mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 20 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, be developing solvent, launch, take out, dry that spray earlier is with 5% ferric chloride alcoholic solution at 15: 5: 3 with n-butyl alcohol-glacial acetic acid-water, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle; It is clear or put under the ultra-violet lamp 365nm and inspect to speckle colour developing to spray with vanillin sulfuric acid solution after heat wind again, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle.

Embodiment 10: the liquid chromatograph discrimination method of peoniflorin in the pharmaceutical composition

It is an amount of to get prescription liquid drugs injection of the present invention, is dissolved in water or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Alcoholic solution with the peoniflorin reference substance is contrast, adopts liquid chromatography, and chromatographic column is a filler with eight alkyl silane bonded silica gels, and 70%: 30% acetonitrile-water is a mobile phase, and the detection wavelength is 235nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 11: ginsenoside Rg in the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more assay among the ginsenoside Re

It is an amount of to get prescription powder injection formulation of the present invention, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, one or more reference substances among the ginsenoside Re methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with external standard method or standard curve method, prescription powder injection formulation of the present invention is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(4) the per unit amount contains the ginsenoside Rg ₁, the ginsenoside Re the limit of summation must not be less than 3.4mg.

Embodiment 12: the assay of total saponins in the pharmaceutical composition

It is an amount of to get prescription powder injection formulation of the present invention, puts in the 10ml measuring bottle, adds water to scale, shake up, precision is measured 0.6ml, to 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 cooling 2 minutes, precision added glacial acetic acid to 25ml 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, wavelength place at 547nm measures trap, calculate with external standard method or standard curve method, prescription powder injection formulation of the present invention 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 6mg.

Embodiment 13: content of paeoniflorin is measured in the pharmaceutical composition

It is an amount of to get prescription powder injection formulation of the present invention, adds dissolve with methanol or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Methanol solution with the peoniflorin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% formic acid solution was a mobile phase in 40%: 60%, and the detection wavelength is 230nm; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains peoniflorin must not be less than 20mg.

Embodiment 14: ginsenoside Rg in the pharmaceutical composition ₁, ginsenoside Rb ₁, one or more assay among the ginsenoside Re

It is an amount of to get prescription dispersible tablet formulation of the present invention, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, one or more reference substances among the ginsenoside Re alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, methanol-2.5% glacial acetic acid aqueous solution was a mobile phase in 25%: 75%, gradient elution, solvent ratios is from 0 minute to 30 minutes, the ratio of acetonitrile is 25%, from 30 minutes to 50 minutes, the ratio of acetonitrile rises to 30% by 25%, and from 50 minutes to 70 minutes, the ratio of acetonitrile was 30%, from 70 minutes to 90 minutes, the ratio of acetonitrile rose to 50% by 30%; Flow velocity is 1.8ml/min, and the detection wavelength is 200nm, 50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, dispersible tablet formulation is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(5) the per unit amount contains the ginsenoside Rg ₁Limit must not be less than 0.8mg;

(6) the per unit amount limit that contains the ginsenoside Re must not be less than 0.4mg;

(7) the per unit amount contains ginsenoside Rb ₁Limit must not be less than 0.5mg;

(8) the per unit amount contains the ginsenoside Rg ₁, the ginsenoside Re the limit of summation must not be less than 1.7mg.

Embodiment 15: the assay of total saponins in the pharmaceutical composition

It is an amount of to get prescription hydro-acupuncture preparation of the present invention, puts in the measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured in right amount, puts in the measuring bottle, water bath method takes out immediately, and precision adds 18% vanillin-glacial acetic acid solution 2ml, perchloric acid 0.2ml shakes up, and heating is 5 minutes in 80 ℃ of water-baths, take out, with the flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with the ginsenoside Rg ₁Or ginsenoside Rb ₁Or ginsenoside Re or ginsenoside Rf be reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with external standard method or standard curve method at the wavelength place of 547 ± 10nm, hydro-acupuncture preparation 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 ginsenoside Re or ginsenoside Rf's meter, must not be less than 3mg.

Embodiment 16: content of paeoniflorin is measured in the pharmaceutical composition

It is an amount of to get prescription dispersible tablet formulation of the present invention, is dissolved in water or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Alcoholic solution with the peoniflorin reference substance is contrast, adopts liquid chromatography, and chromatographic column is a filler with eight alkyl silane bonded silica gels, and 70%: 30% acetonitrile-water is a mobile phase, and the detection wavelength is 235nm; Calculate with one point external standard method or standard curve method, dispersible tablet formulation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains peoniflorin must not be less than 10mg.

Embodiment 17: adopting liquid chromatography test Radix Codonopsis composition characteristics is main finger printing:

(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 methanol and make the solution that every 1ml contains 50mg, filters, and gets subsequent filtrate as need testing solution;

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

Embodiment 18: adopting liquid chromatography test Radix Codonopsis composition characteristics is main finger printing:

(1) preparation of need testing solution: it is an amount of to get prescription liquid drugs injection of the present invention, is dissolved in water or dilutes, and 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 codonopsis pilosula, comprise in lobetyolin, the atractylenoide one or both, the water dissolved dilution is to suitable concn, as object of reference solution;

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

(6) with the finger printing of liquid drugs injection 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 liquid drugs injection, should be 0.80～1.00;

II. in the hydro-acupuncture preparation finger printing, non-total peak area must not surpass 10% of total peak area;

Embodiment 19: the thin layer chromatography discrimination method of lobetyolin in the pharmaceutical composition

It is an amount of to get prescription powder injection formulation of the present invention, add methanol 25ml, supersound process 30 minutes filters, the filtrate evaporate to dryness, residue adds water 2ml makes dissolving, puts in the C18 solid phase extraction column (500mg is with methanol, each the 10ml prewashing of 20% methanol), use 20% methanol, each 5ml eluting of methanol successively, collect meoh eluate, be concentrated into 1ml, as need testing solution; Other gets the Radix Codonopsis control medicinal material, shines medical material solution in pairs with legal system; It is an amount of to get lobetyolin's reference substance again, adds methanol and makes every 1ml and contain 1mg solution, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw need testing solution 6 μ l, reference substance solution 2 μ l, put respectively on same high-efficient silica gel G lamellae, with n-butyl alcohol-glacial acetic acid-water is developing solvent at 7: 1: 0.5, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ were heated 5 minutes, and put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

Embodiment 20: the liquid chromatograph discrimination method of lobetyolin in the pharmaceutical composition:

It is an amount of to get prescription powder injection formulation of the present invention, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 21: the thin layer chromatography discrimination method of lobetyolin in the pharmaceutical composition:

It is an amount of to get prescription drop pill of the present invention, is dissolved in water, and uses ethyl acetate extraction, and extract is concentrated into dried, the residue dissolve with ethanol, and as need testing solution, other gets one or both preparation contrast solutions in Radix Codonopsis control medicinal material, the lobetyolin; The preparation of control medicinal material solution: get the Radix Codonopsis control medicinal material, filter behind the extracting in water, the filtrate evaporate to dryness, residue is used ethyl acetate extraction after with water dissolution, and extract is concentrated into dried, the residue dissolve with ethanol, as need testing solution, the preparation of reference substance solution: get lobetyolin's reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 25 μ l of above-mentioned solution, put respectively on same silica GF254 lamellae, n-butyl alcohol-formic acid-water is developing solvent at 20: 5: 2, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and 100 ℃ were heated 25 minutes, and put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

Embodiment 22: the liquid chromatograph discrimination method of lobetyolin in the pharmaceutical composition:

It is an amount of to get prescription dispersible tablet of the present invention, puts in the measuring bottle, adds dissolve with ethanol and is diluted to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and chromatographic column is a filler with eight alkyl silane bonded silica gels, and acetonitrile-0.25% phosphate aqueous solution detects wavelength 265nm, 45 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 23: the assay of lobetyolin in the pharmaceutical composition

It is an amount of to get prescription powder injection formulation of the present invention, puts in the measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, powder injection formulation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains lobetyolin must not be less than 0.1mg.

Embodiment 24: the assay of lobetyolin in the pharmaceutical composition

It is an amount of to get prescription hydro-acupuncture preparation of the present invention, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and chromatographic column is a filler with the dialkyl silane bonded silica gel, and methanol-0.08% phosphate aqueous solution is a mobile phase at 60%: 40%, and the detection wavelength is 263nm, 40 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, hydro-acupuncture preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains lobetyolin must not be less than 0.05mg.

Claims

1, a kind of method of quality control of the pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin, 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 pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin of claim 1, it is characterized in that: this method adopt liquid chromatography test with Radix Ginseng Rubra or constituent of ginseng be characterized as main finger printing, based on the finger printing of Radix Codonopsis composition characteristics:

3, according to the method for quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin of claim 2, it is characterized in that: this method adopt liquid chromatography test with Radix Ginseng Rubra or constituent of ginseng be characterized as main finger printing, based on the finger printing of Radix Codonopsis composition characteristics:

4, according to the method for quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin of claim 1, it is characterized in that: the discrimination method of described pharmaceutical composition comprises following all or part of content:

It is an amount of to get pharmaceutical composition 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 Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, among the ginsenoside Re, ginsenoside Rf one or more, the preparation contrast solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, add the chloroform reflux, extract,, discard chloroform liquid, 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, one or more reference substances among the ginsenoside Re, ginsenoside Rf, 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;

It is an amount of to get pharmaceutical composition 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, one or more reference substances among the ginsenoside Re methanol or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is that filler 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 with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 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 pharmaceutical composition 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 peoniflorin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution or 0.005mol/L～2mol/L sodium dihydrogen phosphate or 0.005mol/L～2mol/L potassium dihydrogen phosphate 5%～95%: 95%～5% is a 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;

It is an amount of to get pharmaceutical composition to be measured, puts in the measuring bottle, adds methanol or ethanol or mobile phase to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with lobetyolin's reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～0.5% phosphate aqueous solution 5%～80%: 95%～20% are mobile phase, the detection wavelength is one or several in 200～410nm scope, in 20～50 ℃ of scopes of column temperature; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

5, according to the method for quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin of claim 4, it is characterized in that: the discrimination method of described pharmaceutical composition comprises following all or part of content:

6, according to the method for quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin of claim 1, it is characterized in that: the method for testing of described pharmaceutical composition content should comprise following all or part of content:

It is an amount of to get pharmaceutical composition 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 ₁The methanol of one or more reference substances among the ginsenoside Re or alcoholic solution are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, pharmaceutical composition 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 b, the pharmaceutical composition:

Content of paeoniflorin is measured in c, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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 peoniflorin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution or 0.005mol/L～2mol/L sodium dihydrogen phosphate or 0.005mol/L～2mol/L potassium dihydrogen phosphate 5%～95%: 95%～5% is a 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 peoniflorin must not be less than 10mg;

The assay of lobetyolin in d, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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; Methanol solution with lobetyolin's reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～0.5% phosphate aqueous solution 5%～80%: 95%～20% are mobile phase, the detection wavelength is one or several in 200～410nm scope, in 20～50 ℃ of scopes of column temperature; 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 lobetyolin must not be less than 0.05mg.

7, according to the method for quality control of the described pharmaceutical composition made from Radix Ginseng or Radix Ginseng Rubra or Radix Codonopsis, peoniflorin of claim 6, it is characterized in that: the method for testing of described pharmaceutical composition content is following one or more methods:

The assay of total saponins in b, the pharmaceutical composition:

Content of paeoniflorin is measured in c, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition 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 solution with the peoniflorin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% formic acid solution was a mobile phase in 40%: 60%, and the detection wavelength is 230nm; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains peoniflorin must not be less than 20mg;

The assay of lobetyolin in d, the pharmaceutical composition:

8, according to the method for quality control of claim 6 or 7 described pharmaceutical compositions, it is characterized in that: the assay result of described pharmaceutical composition, can survey composition except that adjuvant, the ginsenoside Rg in its quality standard ₁, ginsenoside Rb ₁, all or part of kind among ginsenoside Re, peoniflorin, total saponins, lobetyolin, atractylenoide etc. total content account for more than 25%.