CN101158670A

CN101158670A - Quality control method of pharmaceutical composition

Info

Publication number: CN101158670A
Application number: CNA2006101529770A
Authority: CN
Inventors: 于文风
Original assignee: Qiyuanyide Medicines Institute Beijing
Current assignee: Qiyuanyide Medicines Institute Beijing
Priority date: 2005-09-22
Filing date: 2006-09-22
Publication date: 2008-04-09

Abstract

The invention relates to a quality control method of the medicine composition made from breviscapinun and paeoniflorin. The quality control method comprises a breviscapinun in a composition preparation, a fingerprint atlas test method of the paeoniflorin, a distinction test method of the paeoniflorin and a content determination method of the paeoniflorin. The invention provides a reliably, stable, a bran-new method for the quality control of the composition preparation so as to lead the control of the composition preparation to be stricter and more reasonable and the quality is more stable. At the same time, the invention also provides a reliable and stable examination method for a big production, and a digitized explanation for the guarantee of the validity and security of the drug which a patient takes.

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 the technical field of medicine.

Background technology

This pharmaceutical composition is that raw material is made with Breviscapinun, Paeoniflorin, it is characterized in that: calculate according to parts by weight, it is made with extra care and is formed by 1～10 part of Breviscapinun, 1～100 part of warp of Paeoniflorin; This pharmaceutical composition is that comprehensive traditional Chinese medical theory and the assembly of modern medicine and pharmacology principle form, have and activate blood circulation and disperse blood clots, promoting blood circulation, activating collaterals, improve blood circulation and metabolism effect, be applicable to the hemiplegia of apoplexy syndrome of static blood blocking collaterals, facial paralysis, speech is stuttering puckery, tongue is thin white or thin white greasy, deep thready pulse etc., and cerebral infarction is plugged with the above-mentioned symptom of opinion, except that being used for the treatment of cardiovascular and cerebrovascular diseases such as coronary heart disease, angina pectoris, arrhythmia cordis, cerebral thrombus, senile dementia, also be used for diseases such as hepatorenal syndrome, heart and lung diseases, diabetes and complication thereof clinically.Cardiovascular and cerebrovascular disease such as coronary heart disease, cerebral thrombus, senile dementia 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, the incidence of disease of such disease has and increases trend year by year in recent years, and in, young patient constantly increases, and become common disease, the frequently-occurring disease of harm China people ' s health.Quality for better this pharmaceutical composition of control guarantees the security of medication, and better guidance production makes technology controlling and process rationally strict more, makes consumer's energy full appreciation product quality, and the applicant has studied the method for quality control of this pharmaceutical composition.The applicant not only provides the discrimination method and the content assaying method of characteristic components such as scutellarin, Paeoniflorin, has more set up the technical scheme of finger-print, and its material group integral body is controlled, and effectively characterizes its quality on the whole.That traditional Chinese medicine fingerprint is meant is common in certain Chinese crude drug or the Chinese patent drug, have distinctive certain class or the chromatogram of number constituents or the collection of illustrative plates of spectrum.In present stage, the most effective constituent of Chinese medicine does not have under the clear and definite situation, and finger-print has useful effect for the quality of effective this medicine composition of control.Discriminating, assay, finger-print use in conjunction, broad covered area, high specificity is the outstanding substantive distinguishing features of this method of quality control.

Summary of the invention

The objective of the invention is to: a kind of method of quality control of pharmaceutical composition is provided, and 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 security of medication, can better instruct production, make controlling of production process rationally strict more, make consumer's energy full appreciation product quality.

The present invention constitutes like this: a kind of method of quality control of the pharmaceutical composition made from Breviscapinun, Paeoniflorin, and it is characterized in that: this method comprises following all or part of content:

(1) based on the fingerprint test method of all or part of composition characteristics in Breviscapinun, the Paeoniflorin;

(2) the differential test method of one or both compositions in scutellarin, the Paeoniflorin;

(3) content test method of one or both compositions in scutellarin, the Paeoniflorin.

The method of quality control of the described pharmaceutical composition made from Breviscapinun, Paeoniflorin is characterized in that this method comprises all or part of content of following finger-print test:

(1) preparation of need testing solution: it is an amount of to get pharmaceutical composition to be measured, is solvent with in water or methyl alcohol or the ethanol one or more, extracts or dilution or dissolving the preparation need testing solution;

(2) preparation of object of reference solution: get scutellarin or Paeoniflorin, one or more in water or methyl alcohol or the ethanol are solvent, are dissolved to suitable concn, as object of reference solution;

(3) chromatographic condition: chromatographic column adopting alkyl silane bonded silica gel is a filler: mobile phase A is 50～100% acetonitriles or 50～100% methyl alcohol, Mobile phase B is 0.005mol/L～0.3mol/L sodium dihydrogen phosphate or 0.005mol/L～0.3mol/L potassium dihydrogen phosphate or 0.1%～3% glacial acetic acid or 0.1%～3% formic acid or 0.03%～1% phosphoric acid solution, gradient elution, mobile phase A ratio variation range is 12～85%, flow velocity is that 0.5～1.5ml/min, detection wavelength are one or several in the 190-400nm scope, and column temperature is at 20～50 ℃;

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Breviscapinun, the root of herbaceous peony or radix paeoniae rubrathe composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 2～8;

(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-print of Breviscapinun, the root of herbaceous peony or radix paeoniae rubrathe composition characteristics, the finger-print of preparation testing sample;

(6) finger-print of pharmaceutical composition to be measured, in should meeting the following requirements partly or entirely:

I. the similarity of the finger-print of pharmaceutical composition to be measured and standard finger-print should be 0.80～1.00;

II. non-total peak area must not surpass 10% of total peak area;

III. the ratio 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 and standard finger-print compare, and relative deviation must not surpass ± 20%;

IV. unimodal area accounts for total peak area than greater than 20% peak in the total peak, must not surpass ± 30% with the ratio relative deviation of standard finger-print respective peaks peak area; Unimodal area accounts for total peak area than greater than 10% less than 20% peak in the total peak, must not surpass ± 35% with the ratio relative deviation of standard finger-print respective peaks peak area; Unimodal area accounts for total peak area than greater than 5% less than 10% peak in the total peak, must not surpass ± 40% with the ratio relative deviation of standard finger-print respective peaks peak area.

The method of quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony is characterized in that this method comprises all or part of content of following finger-print test:

(1) preparation of need testing solution: it is an amount of that precision takes by weighing pharmaceutical composition to be measured, and the solution that every 1ml contains 1mg is made in thin up or dissolving, filters, promptly;

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

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

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Breviscapinun, the root of herbaceous peony or radix paeoniae rubrathe composition characteristics; Accurate respectively object of reference solution and each 10 μ l of need testing solution of drawing, inject liquid chromatograph 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 2～6;

I. the similarity of the finger-print of pharmaceutical composition to be measured and standard finger-print should be 0.90～1.00;

II. non-total peak area must not surpass 5% of total peak area;

III. the ratio 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 and standard finger-print compare, and relative deviation must not surpass ± 10%;

IV. unimodal area accounts for total peak area than greater than 20% peak in the total peak, must not surpass ± 20% with the ratio relative deviation of standard finger-print respective peaks peak area; Unimodal area accounts for total peak area than greater than 10% less than 20% peak in the total peak, must not surpass ± 25% with the ratio relative deviation of standard finger-print respective peaks peak area; Unimodal area accounts for total peak area than greater than 5% less than 10% peak in the total peak, must not surpass ± 30% with the ratio relative deviation of standard finger-print respective peaks peak area.

The method of quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony is characterized in that this method comprises all or part of content in the following differential test method:

The thin-layer chromatography discrimination method of scutellarin in a, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition to be measured, adds ethanol or methyl alcohol or normal butyl alcohol and extracts, and filtration or centrifugal is got filtrate or supernatant as need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol or ethanol and makes the reference substance solution that every 1ml contains 1mg; The test of employing thin-layered chromatography, it is an amount of to draw above-mentioned solution respectively, puts respectively on same silica gel thin-layer plate or polyamide film, and with methyl alcohol or ethanol-ethyl acetate or ethyl formate or butyl acetate-formic acid or acetate=5～12: be developping agent at 0.5～5: 0.02～3, launch, take out, dry, spray is to contain aluminium choride or ferric trichloride developer, toast or do not toast, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, should show the same color spot;

The liquid chromatography discrimination method of scutellarin in b, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition to be measured, is solvent with in methyl alcohol or ethanol or the water one or more, extracts or dilution or dissolving, is made as need testing solution; Prepare reference substance solution with the scutellarin reference substance, adopt liquid phase chromatography, chromatographic column is a filling agent with the alkyl silane bonded silica gel, with methyl alcohol or acetonitrile-water or 0.2%～3% glacial acetic acid aqueous solution or 0.2%～3% aqueous formic acid or 0.01%～1% phosphate aqueous solution or 0.005mol/L～0.3mol/L sodium dihydrogen phosphate or 0.005mol/L～0.3mol/L potassium dihydrogen phosphate=16～70: 84～30 is moving phase, detects wavelength and be among 200～410nm one or more; Employing liquid phase chromatography test is drawn need testing solution respectively with reference substance solution is an amount of, injects liquid chromatograph, tests, and in the test sample chromatogram, answers tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;

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

It is an amount of to get pharmaceutical composition to be measured, adds in ethanol or methyl alcohol or normal butyl alcohol or the water one or more for solvent extraction, the preparation need testing solution; Get the Paeoniflorin reference substance, make reference substance solution; The test of employing thin-layered chromatography, it is an amount of to draw above-mentioned contrast solution and test solution respectively, point is on same silica gel thin-layer plate, with methenyl choloride or methylene chloride-ethyl acetate or ethyl formate or butyl acetate-methyl alcohol or ethanol-formic acid or acetate=2～8: be developping agent at 0.5～5: 0.5～5: 0.01～4, launch, take out, dry, successively or spray with the developer that contains ferric trichloride or developer that contains aluminium choride or vitriolated developer respectively or contain in the developer of vanillic aldehyde one or more, in the test sample chromatogram, with contrast chromatogram corresponding position on, should show the same color spot;

The liquid chromatography discrimination method of Paeoniflorin in d, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition to be measured, adds one or more extractions or the dissolving in methyl alcohol or ethanol or the water or is diluted to suitable concn, as need testing solution; Prepare reference substance solution with the Paeoniflorin reference substance, adopt liquid phase chromatography, chromatographic column is a filling agent with the alkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～3% glacial acetic acid aqueous solution or 0.05%～3% aqueous formic acid or 0.001%～1% phosphate aqueous solution or 0.005mol/L～0.3mol/L sodium dihydrogen phosphate or 0.005mol/L～0.3mol/L potassium dihydrogen phosphate=15～62: 85～38 moving phases, detection wavelength are one or several among 200～410nm; Employing liquid phase chromatography test is drawn need testing solution respectively with reference substance solution is an amount of, injects liquid chromatograph, tests, and in the test sample chromatogram, answers tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;

It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, the preparation need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg; The test of employing thin-layered chromatography, draw each 3 μ l of above-mentioned solution, putting on same polyamide membrane with strip tape respectively, is developping agent with methyl alcohol-ethyl acetate-formic acid=7: 2: 1, launches, take out, dry, spray is with 2% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the streak of same color;

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 scutellarin reference substance is contrast, adopts liquid phase chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-0.1% phosphate aqueous solution=50: 50 is a moving phase, and the detection wavelength is 335nm; Draw need testing solution respectively and reference substance solution is an amount of, inject liquid chromatograph, test, in the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;

It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, centrifugal, gets supernatant as need testing solution; The methanol solution that other gets the Paeoniflorin reference substance is product solution in contrast; The test of employing thin-layered chromatography, it is an amount of to draw test solution and contrast solution, puts respectively on same silica gel g thin-layer plate, and with methenyl choloride-ethyl acetate-methyl alcohol-formic acid=6: 1.5: 1.5: 0.5 was developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, it is clear to be heated to the spot colour developing, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction or dilution, filters, and gets subsequent filtrate as need testing solution; Methanol solution with the Paeoniflorin reference substance is a reference substance solution, adopts liquid phase chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-0.1% formic acid solution=40: 60 is a moving phase, and the detection wavelength is 230nm; Draw need testing solution respectively and reference substance solution is an amount of, inject liquid chromatograph, test, in the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.

The method of quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony is characterized in that this method comprises all or part of content of following assay:

The assay of scutellarin in a, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition to be measured, is solvent with in methyl alcohol or ethanol or the water one or more, extracts or dilution or dissolving, is made as need testing solution; Prepare reference substance solution with the scutellarin reference substance, adopt liquid phase chromatography, chromatographic column is a filling agent with the alkyl silane bonded silica gel, with methyl alcohol or acetonitrile-water or 0.2%～3% glacial acetic acid aqueous solution or 0.2%～3% aqueous formic acid or 0.01%～1% phosphate aqueous solution or 0.005mol/L～0.3mol/L sodium dihydrogen phosphate or 0.005mol/L～0.3mol/L potassium dihydrogen phosphate=16～70: 84～30 is moving phase, detects wavelength and be among 200～410nm one or more; Employing liquid phase chromatography test, accurate respectively to draw need testing solution an amount of with reference substance solution, injects liquid chromatograph, tests, and calculates with one point external standard method or calibration curve method, and must not be less than 12mg with what dosage contained scutellarin every day in the pharmaceutical composition;

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

It is an amount of to get pharmaceutical composition to be measured, adds one or more extractions or the dissolving in methyl alcohol or ethanol or the water or is diluted to suitable concn, filters, as need testing solution; Prepare reference substance solution with the Paeoniflorin reference substance, adopt liquid phase chromatography, chromatographic column is a filling agent with the alkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～3% glacial acetic acid aqueous solution or 0.05%～3% aqueous formic acid or 0.001%～1% phosphate aqueous solution or 0.005mol/L～0.3mol/L sodium dihydrogen phosphate or 0.005mol/L～0.3mol/L potassium dihydrogen phosphate=15～62: 85～38 moving phases, detection wavelength are one or several among 200～410nm; Employing liquid phase chromatography test, it is accurate respectively that to draw need testing solution an amount of with reference substance solution, and the injection liquid chromatograph calculates with one point external standard method or calibration curve method, and must not be less than 15mg with what dosage contained Paeoniflorin every day in the pharmaceutical composition;

The assay of scutellarin in a, the pharmaceutical composition:

It is an amount of to get pharmaceutical composition to be measured, and accurate the title decides or measure, and adds methanol extraction or is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution; Methanol solution with the scutellarin reference substance is contrast, adopts liquid phase chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-0.1% phosphate aqueous solution=50: 50 is a moving phase, and the detection wavelength is 335nm; Respectively accurate to draw need testing solution an amount of with reference substance solution, injects liquid chromatograph, tests, and calculates with one point external standard method, and must not be less than 25mg with what dosage contained scutellarin every day in the pharmaceutical composition;

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

It is an amount of to get pharmaceutical composition to be measured, and accurate the title decides or measure, and 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 Paeoniflorin reference substance is contrast, adopts liquid phase chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-0.1% formic acid solution=40: 60 is a moving phase, and the detection wavelength is 230nm; Respectively accurate to draw need testing solution an amount of with reference substance solution, injects liquid chromatograph, tests, and calculates with one point external standard method, and must not be less than 30mg with what dosage contained Paeoniflorin every day in the pharmaceutical composition;

The method of quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony, it is characterized in that: the assay result of described pharmaceutical composition, calculate according to percentage by weight, the total amount of scutellarin and Paeoniflorin accounts for deduction auxiliary material and outer more than 50% of total solid of moisture in the preparation.

Compared with prior art, the present invention's quality of the pharmaceutical composition made with Breviscapinun, Paeoniflorin of perfect control more.The traditional 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 controls the quality of injection comprehensively from finger-print, discriminating and three aspects of assay.Because the chemical constitution phase mutual interference behind preparations shaping in the drug regimen raw material all can affect greatly discriminating, assay, finger-print.Originally once be applicable to discriminating, assay or the fingerprint test method of single raw material, be used to test contain this raw material pharmaceutical composition the time often can't implement.Therefore, the method for quality control of pharmaceutical composition must be done with bigger improvement on the basis of former single raw material correlation method, or abandons former method and set up diverse new method.That is to say that quality control method of the present invention is not to be the simple superposition of single raw material quality control method in the composition, but after a large amount of experimental studies, invent.

And have only the condition of the present invention of employing, just can obtain desirable discriminating, assay and finger-print Quality Control effect.

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 Breviscapinun, Paeoniflorin or Paeoniflorin, and method precision, stability are all higher.

In experimental example 1 injection sterile powder based on the preparation of the finger-print of Breviscapinun, Paeoniflorin composition characteristics

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 filling agent, has tried out Inertsil ODS-3 (250mm * 4.6mm, 5 μ m) Kromasil C respectively ₁₈(200mm * 4.6mm, 5 μ m), Diamonsil C ₁₈The chromatographic column of (250mm * 4.6mm, 5 μ m) three kinds of trades mark, the result shows that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, wherein DiamonsilC ₁₈The chromatographic column separating effect is best, and post is imitated the highest.So select Diamonsil C for use ₁₈(250mm * 4.6mm, 5 μ m) are the experimental study post.

2. the selection of moving phase:

Investigated (1) acetonitrile-water (18: 82) in the research process respectively, (2) methanol-water (40: 60); (3) methyl alcohol-0.05moL/L sodium dihydrogen phosphate (10: 90); (4) acetonitrile solution-0.5% phosphoric acid (gradient elution); (5) methanol solution-0.08moL/L potassium dihydrogen phosphate (gradient elution); (6) acetonitrile solution-1% glacial acetic acid 6 kinds of flow phase system such as (gradient elutions); The result shows under moving phase (1) condition that the peak shape hangover separates bad; Under moving phase (2) condition, the peak shape hangover, peak and overlap of peaks are more; Under moving phase (3) condition, 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 moving phase (4), (5), (6) condition, 50～100% acetonitriles or 50～100% methyl alcohol-0.005mol/L～0.3mol/L sodium dihydrogen phosphate or 0.005mol/L～0.3mol/L potassium dihydrogen phosphate or 0.1%～3% glacial acetic acid or 0.1%～3% formic acid or 0.03%～1% phosphoric acid solution=12～85: in 88～15 scopes, gradient elution can obtain preferable effect; Top condition is: mobile phase A is 80% acetonitrile, and Mobile phase B is 0.1% phosphoric acid, gradient elution, from 0 minute to 80 minutes, the ratio of mobile phase A rose to 70% by 15%, and flow velocity is 1.0ml/min, under this condition, peak shape is good, and it is fast and fully and be evenly distributed to go out the peak.

3. detection wavelength determination:

Under above-mentioned optimal flow phase condition, investigated the chromatographic peak situation under 190～410nm respectively, the result shows that 230nm can take into account kurtosis, degree of separation and the baseline of each composition chromatographic peak when detecting, best results is so select 230nm to detect wavelength for this product finger-print.

4. instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved LC-10Avp high performance liquid chromatograph for use, WML-2010 chromatographic work station, 40 ℃ of column temperatures.

5. the preparation of need testing solution:

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

6. the preparation of object of reference solution:

It is an amount of to take by weighing the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.15mg approximately, shakes up, promptly.

7. finger-print and technical parameter:

7 formulate standard finger-print according to 10 batch samples, and it is as follows to formulate the fingerprint image standard of pharmaceutical composition to be measured:

I. pharmaceutical composition to be measured finger-print and the similarity of standard finger-print, should be 0.90～1.00;

II. non-total peak area must not surpass 5% of total peak area;

III. the ratio 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%;

IV. unimodal area accounts for total peak area than greater than 20% peak in the total peak, must not surpass ± 20% with the ratio relative deviation of standard finger-print respective peaks peak area; Unimodal area accounts for total peak area than greater than 10% less than 20% peak in the total peak, must not surpass ± 25% with the ratio relative deviation of standard finger-print respective peaks peak area; Unimodal area accounts for total peak area than greater than 5% less than 10% peak in the total peak, must not surpass ± 30% with the ratio relative deviation of standard finger-print respective peaks peak area;

The thin-layer chromatography discrimination method of scutellarin in experimental example 2 parenteral solutions:

For the feature of outstanding oil lamp element, selected scutellarin as its feature spot, have only the interference of getting rid of compositions such as Paeoniflorin in the preparation, could obtain desirable chromatogram effect.The key factor of thin-layered chromatography effect quality is the composition of unfolding condition, particularly developping agent.Therefore, experiment sieving multiple unfolding condition, part unfolding condition and result are as follows:

The thin-layer chromatography discrimination method of scutellarin in the parenteral solution

Condition question

Benzene-ethyl formate-formic acid (5: 3: 0.5) silica gel g thin-layer plate separates unintelligible

Ethyl acetate-benzene-acetate (5: 4: 3) polyamide film separates unintelligible, and feminine gender has interference

Ethyl acetate-benzene-formic acid (8: 4: 3) silica gel g thin-layer plate separates unintelligible

Chloroform-ethyl acetate-acetone (7: 2: 4) silica gel H thin layer plate separates unintelligible

Toluene-ethyl acetate (8: 1) silica gel H thin layer plate separates unintelligible

Ethanol-acetone-formic acid (10: 3: 1) polyamide film reference substance is expanded to the forward position

It is clear that methyl alcohol-ethyl acetate-formic acid (7: 2: 1) polyamide film separates, and the Rf value is moderate, and negative do not have

Disturb

Through screening, determined best unfolding condition: be stationary phase with the polyamide film, with methyl alcohol: ethyl acetate: formic acid (7: 2: 1) be developping agent, and with this understanding, the Rf value of scutellarin is moderate, and it is clear to separate with other spot, and feminine gender is noiseless.

The thin-layer chromatography discrimination method of experimental example 3 concentrated solution for injection Paeoniflorins:

Test and Selection Paeoniflorin be the feature spot, but have only the interference of getting rid of compositions such as scutellarin, could obtain desirable chromatogram effect.The key factor of thin-layered chromatography effect quality is the composition of unfolding condition, particularly developping agent.Therefore, experiment sieving multiple unfolding condition, part unfolding condition and result are as follows:

The thin-layer chromatography of concentrated solution for injection Paeoniflorin, gallic acid is differentiated

Condition question

Methylene chloride-ethyl acetate-methyl alcohol (9: 6: 5) silica gel g thin-layer plate reference substance is expanded to the forward position

Ethyl acetate-methyl alcohol-formic acid (7: 6: 5) silica G F ₂₅₄The thin layer plate feminine gender has interference

Methenyl choloride-ethanol-glacial acetic acid (7: 2: 2) silica gel H thin layer plate separates unintelligible, and feminine gender has interference

Methenyl choloride-ethyl acetate-formic acid (7: 1: 2) silica gel g thin-layer plate separates unintelligible

Acetone-ethyl acetate-glacial acetic acid (7: 1: 2) silica gel H thin layer plate separates unintelligible,

(8: 3: 3: 2) the silica gel H thin layer plate separated unintelligible normal hexane-butyl acetate-ethanol-formic acid

(6: 1.5: 1.5: 0.5) separation of silica G thin layer was clear, and the Rf value is moderate, and negative do not have for methenyl choloride-ethyl acetate-methyl alcohol-formic acid

Plate disturbs

Through screening, determined best unfolding condition: be stationary phase with the silica gel g thin-layer plate, with methenyl choloride: ethyl acetate: methyl alcohol: formic acid (6: 1.5: 1.5: 0.5) be developping agent, with this understanding, the Rf value of Paeoniflorin, gallic acid is moderate, it is clear to separate with other spot, negative noiseless.

The assay of scutellarin in experimental example 4 injection sterile powders

1 instrument and reagent

1.1 key instrument:

High performance liquid chromatograph LC-2010AHT SHIMADZU

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

Electronic analytical balance BP211D SARTORIUS

Supersonic wave cleaning machine KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.

1.2 reagent:

Scutellarin Nat'l Pharmaceutical ﹠ Biological Products Control Institute

Methyl alcohol is analyzed pure Beijing Chemical Plant

Acetonitrile chromatographically pure Di Ma company

The pure Beijing of phosphate analysis chemical reagents corporation

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

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

4 chromatographic conditions

Chromatograph: SHIMADZU LC-2010AHT;

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

Moving phase: methyl alcohol-0.1% phosphoric acid (50: 50);

Flow velocity: 1.0ml/min;

Column temperature: 30 ℃;

Sample size: 10 μ l;

Detect wavelength: 335nm.

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

The about 20mg of this product powder is got in the preparation of need testing solution, the accurate title, decide, put in the 50ml measuring bottle, add an amount of sonicated of methyl alcohol (power 250W, frequency 33KHz) 5min, take out, put to room temperature, it is fixed to scale to add methyl alcohol, shakes up, filter with miillpore filter (0.45 μ m), get subsequent filtrate as need testing solution.

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

Obtain scutellarin reference substance, test sample chromatogram according to above-mentioned chromatographic condition, its number of theoretical plate n is all greater than 5000, and the scutellarin chromatographic peak separates clear complete with close peak, and degree of separation is all greater than 1.5, and solvent is noiseless.

The about 40mg of this product powder (totally 6 parts) is got in the selection of 5 extraction times, and accurate the title decides, and splits in the 25ml measuring bottle, it is an amount of to add methyl alcohol, respectively sonicated (power 250W, frequency 33KHz) 5,10,20min, take out, put, add methyl alcohol to scale to room temperature, shake up, filter with miillpore filter (0.45 μ m), the accurate subsequent filtrate 10 μ l that draw inject liquid chromatograph, measure, promptly.

Extraction time is investigated

Extraction time (min) content (mg/ bottle)

5 27.61

10 27.54

20 27.63

The result shows that sonicated 5min can extract fully.

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

Scutellarin standard curve determination result

Numbering scutellarin amount (μ g) conversion back (μ g) peak area

1 0.0000 0.0000 0

2 0.1956 0.1886 603722

3 0.3912 0.3771 1224078

4 0.5868 0.5657 1835374

5 0.7824 0.7542 2505678

6 0.9780 0.9428 3038182

Regression equation: Y=3259091.50x-1830.06

Related coefficient: γ=0.9999

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

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

The experiment of 7 precision is accurate draws with a scutellarin reference substance solution 10 μ l, injects liquid chromatograph, measures 5 times, investigates reference substance solution precision, and measurement result sees the following form.

The precision test

Test number (TN) 12345 mean value RSD (%)

Peak area 1,572,015 1,570,202 1,566,206 1,559,384 1,557,017 1,564,965 0.42

The result shows that reference substance solution precision is good.

8 need testing solution stability experiments

The same need testing solution 10 μ l of accurate absorption inject liquid chromatograph, measure at 0,2,4,8,24 hour sample introduction respectively.

The need testing solution stability experiment

Times (h) 0248 24 average RSD

Content (mg/ bottle) 27.46 26.94 26.88 27.19 26.07 26.91 1.94

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

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

Repeated experiment

Number 12345 mean value RSD (%)

Content (mg/ bottle) 27.35 27.12 27.64 26.95 27.05 27.22 1.01

The application of sample absorption method is adopted in the experiment of 10 average recoveries; get the about 10mg of this product powder (totally 6 parts); the accurate title decides; split in the 100ml measuring bottle; accurate respectively scutellarin reference substance solution (C=1.107mg/ml) 1.6ml, 2.0ml and the 2.4ml (each two parts) of adding; add an amount of sonicated of methyl alcohol (power 250W, frequency 33KHz) 5min, take out; put to room temperature; add methyl alcohol to scale, shake up, use miillpore filter (0.45 μ m) to filter; the accurate subsequent filtrate 10 μ l that draw; inject liquid chromatograph, mensuration, promptly.(content of scutellarin is in the pharmaceutical composition: 22.85%.)

The average recovery experiment

Test sample claims the pure product scutellarin addition conversion back measured value recovery in the sample test sample

Numbering

Amount (mg) amount (mg) is (mg) (mg) (%) (mg)

1 10.08 2.3033 1.7712 1.7074 3.9615 97.12

2 9.44 2.1570 1.7712 1.7074 3.8071 96.64

3 11.26 2.5729 2.214 2.1343 4.6182 95.83

4 10.94 2.4998 2.214 2.1343 4.5549 96.29

5 12.05 2.7534 2.6568 2.5612 5.2409 97.12

6 10.24 2.3398 2.6568 2.5612 4.8150 96.64

Average recovery rate=96.61%, RSD=0.51%

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

Sample size is measured

Lot number scutellarin content (mg/ bottle)

1 27.12

2 26.54

3 29.73

Paeoniflorin content is measured in experimental example 5 injection sterile powders

1 instrument and reagent

Instrument: JASCO 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

Methyl alcohol is analyzed pure Beijing Chemical Plant

Sodium dihydrogen phosphate is analyzed pure Beijing chemical reagents corporation

2 methods and result

2.1 chromatographiccondition

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

Moving phase: methyl alcohol-0.1% formic acid (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 Paeoniflorin reference substance, sample chromatogram figure (seeing accompanying drawing 12,13), number of theoretical plate n is all greater than 3000.

2.3 the selection of measuring wavelength is through UV scanning, Paeoniflorin 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 Paeoniflorin reference substance 10mg of phosphorus pentoxide vacuum drying apparatus, puts in the 100ml measuring bottle, adds methyl alcohol and makes dissolving in right amount and be diluted to scale, shakes up, and promptly gets (containing Paeoniflorin 0.1mg among every 1ml).

2.5 5 bottles of this product are got under the content uniformity item in the preparation of need testing solution, get content, mixing is got about 0.1g, the accurate title, decide, and puts in the 25ml measuring bottle, and it is an amount of to add methyl alcohol, sonicated (power 250W, frequency 33KHZ) made dissolving in 10 minutes, take out, put coldly, be diluted to scale with methyl alcohol, shake up, filter, precision is measured subsequent filtrate 1ml, put in the 10ml measuring bottle, add methyl alcohol and be diluted to scale, shake up, filter with miillpore filter (0.45um), get subsequent filtrate, promptly.

2.6 negative control test for investigating the mensuration whether other auxiliary material disturbs Paeoniflorin, except that Paeoniflorin or Paeoniflorin, takes by weighing other auxiliary material 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 Paeoniflorin reference substance 11.48mg of phosphorus pentoxide vacuum drying apparatus, put in the 10ml measuring bottle, adding methyl alcohol 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 methyl alcohol, 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 ordinate, and Paeoniflorin amount (ug) is horizontal ordinate mapping, drawing standard curve.

Regression equation is Y=1270296.82X+2219.70

γ＝0.9999

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

The standard curve determination data

Numbering Paeoniflorin amount (ug) peak area

1 0.4592 565516.5

2 0.9184 1171490

3 1.3776 1783885

4 1.8368 2344300

5 2.2960 2895713

The accurate need testing solution 10 μ l that draw of 4 need testing solution stability tests inject liquid chromatograph, and every 1 hour replication 1 time, the result shows that within 4 hours need testing solution is stable.

The need testing solution stability test

Average RSD% of times (h) 01234

Paeoniflorin (mg/ bottle) 47.14 45.26 46.94 47.08 46.97 46.66 1.69

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

As follows: the precision test

Number 12345 average RSD%

Peak area 1,374,344 1,376,226 1,378,443 1,375,983 1,376,550 1,376,309 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 determination method item of text need testing solution, and the result is as follows:

Replica test

The average RSD% of test number 12345 X

Paeoniflorin (mg/ bottle) 46.52 45.85 46.23 46.14 45.96 46.14 0.56

7 recovery tests adopt the application of sample absorption method, and precision takes by weighing through dry 36 hours the about 42.46mg of Paeoniflorin of phosphorus pentoxide vacuum drying apparatus, puts in the 25ml measuring bottle, add methyl alcohol and make dissolving in right amount and be diluted to scale, shake up, and promptly get reference substance solution.Precision takes by weighing the about 9mg of this product content, precision is measured reference substance solution 2ml again, puts altogether in the 50ml measuring bottle, adds an amount of sonicated of methyl alcohol and makes dissolving, take out, put coldly, add methyl alcohol, shake up to scale, filter with miillpore filter (0.45um), the accurate subsequent filtrate 10ul that draws injects liquid chromatograph, measures, promptly.(paeoniflorin content is 27.09% in the pharmaceutical composition)

Recovery test

The Chinese herbaceous peony Paeoniflorin adds in the content weighing content

Numbering (mg) glycosides amount (mg) amount (mg) measured value (mg) recovery (%)

1 9.72 2.6331 3.3968 6.0001 99.12

2 9.03 2.4462 3.3968 5.7753 98.01

3 10.01 2.7117 3.3968 6.0287 97.65

4 9.25 2.5058 3.3968 5.8734 99.14

5 10.36 2.8065 3.3968 6.1442 98.26

Average recovery rate=98.44%, RSD=0.68%.

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

The assay of sample

Lot number paeoniflorin content (mg/ bottle)

1 batch 46.25

2 batches 47.92

3 batches 48.24

Embodiment

Embodiment 1 adopts in the liquid chromatography for measuring injection sterile powder and is characterized as main finger-print with Breviscapinun, Paeoniflorin.

(1) preparation of need testing solution: it is an amount of that precision takes by weighing sample powder, adds methyl alcohol and make the solution that every 1ml contains 1mg, shakes up, and uses filtering with microporous membrane, gets subsequent filtrate as need testing solution;

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

(4) be characterized as the formulation of main standard finger-print with Breviscapinun and Paeoniflorin: accurate respectively each the 10 μ l of object of reference solution and need testing solution that draw, inject liquid chromatograph respectively, according to 10 batches of collection of illustrative plates that test sample is measured, the formulation standard finger-print; Retention time with definite object of reference chromatographic peak is a benchmark, calculates the relative retention time of other total chromatographic peak, and in the described standard finger-print, total peak has 2;

(5) with said method as the means of testing that is characterized as main finger-print in the pharmaceutical composition with Breviscapinun, Paeoniflorin, the preparation testing sample finger-print;

(6) finger-print of pharmaceutical composition to be measured should meet the following requirements:

II. non-total peak area must not surpass 5% of total peak area;

Embodiment 2 adopts in the liquid chromatography for measuring concentrated solution for injection and is characterized as main finger-print with Breviscapinun, Paeoniflorin.

(1) preparation of need testing solution: it is an amount of that precision is measured sample, adds 10 times of ethanol dilutions, shakes up, and uses filtering with microporous membrane, gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing scutellarin, adds 95% ethanol and make the solution that every 1ml contains 0.05mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is 50% methyl alcohol, and Mobile phase B is the 0.3mol/L potassium dihydrogen phosphate, gradient elution, and solvent ratios was from 0 minute to 60 minutes, and the ratio of mobile phase A rises to 85% by 20%, and flow velocity is 1.4ml/min, and the detection wavelength is 375 ± 2nm, column temperature is 50 ℃;

(4) be characterized as the formulation of main standard finger-print with Breviscapinun and Paeoniflorin: accurate respectively each the 5 μ l of object of reference solution and need testing solution that draw, inject liquid chromatograph respectively, according to 10 batches of collection of illustrative plates that test sample is measured, the formulation standard finger-print; Retention time with definite object of reference chromatographic peak is a benchmark, calculates the relative retention time of other total chromatographic peak, and in the described standard finger-print, total peak has 6;

II. non-total peak area must not surpass 5% of total peak area;

Embodiment 3 adopts in the liquid chromatography for measuring parenteral solutions finger-print based on Breviscapinun, Paeoniflorin composition characteristics.

(1) preparation of need testing solution: sample thief, with 5 times of methyl alcohol dilutions,, get subsequent filtrate as need testing solution with the filtering with microporous membrane of 0.45 μ m;

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing Paeoniflorin, adds 50% ethanol and make the solution that every 1ml contains 0.05mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is a methyl alcohol, Mobile phase B is 0.1% formic acid solution, gradient elution, and solvent ratios was from 0 minute to 40 minutes, the ratio of mobile phase A rises to 50% by 12%, from 40 minutes to 60 minutes, the ratio of mobile phase A rose to 80% by 50%, from 60 minutes to 65 minutes, the ratio of mobile phase A reduces to 12% by 80%, flow velocity is 0.8ml/min, and the detection wavelength is 190 ± 2nm, and column temperature is 20 ℃;

(4) based on the formulation of the standard finger-print of Breviscapinun and Paeoniflorin composition characteristics: the accurate respectively respectively 10 μ l of object of reference solution and need testing solution that draw, inject liquid chromatograph respectively, according to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print; Retention time with definite object of reference chromatographic peak is a benchmark, calculates the relative retention time of other total chromatographic peak, and in the described standard finger-print, total peak has 2;

(5) with said method as in the pharmaceutical composition based on the means of testing of the finger-print of Breviscapinun, Paeoniflorin composition characteristics, the finger-print of preparation testing sample;

II. non-total peak area must not surpass 10% of total peak area;

Embodiment 4 adopts in the liquid chromatography for measuring injection sterile powders finger-print based on Breviscapinun, Paeoniflorin composition characteristics.

(1) preparation of need testing solution: it is an amount of that precision takes by weighing sample powder, adds 80% methyl alcohol and make the solution that every 1ml contains 0.1mg, shakes up, and with the filtering with microporous membrane of 0.45 μ m, gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing Paeoniflorin, adds 80% methyl alcohol and make the solution that every 1ml contains 0.05mg, as object of reference solution;

(3) chromatographic condition: chromatographic column adopting eight alkyl silane bonded silica gels are filler; Mobile phase A is an acetonitrile, and Mobile phase B is the 0.005mol/L sodium dihydrogen phosphate, gradient elution, and solvent ratios was from 0 minute to 60 minutes, and the ratio of mobile phase A rises to 70% by 12%, and flow velocity is 1.2ml/min, and the detection wavelength is 410 ± 2nm, column temperature is 40 ℃;

(4) based on the formulation of the standard finger-print of Breviscapinun and Paeoniflorin composition characteristics: the accurate respectively respectively 10 μ l of object of reference solution and need testing solution that draw, inject liquid chromatograph respectively, according to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print; Retention time with definite object of reference chromatographic peak is a benchmark, calculates the relative retention time of other total chromatographic peak, and in the described standard finger-print, total peak has 8;

(6) finger-print of pharmaceutical composition to be measured should meet the following requirements: non-total peak area must not surpass 5% of total peak area; The ratio 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 and standard finger-print compare, and relative deviation must not surpass ± 10%; Unimodal area accounts for total peak area than greater than 20% peak in the total peak, must not surpass ± 20% with the ratio relative deviation of standard finger-print respective peaks peak area; Unimodal area accounts for total peak area than greater than 10% less than 20% peak in the total peak, must not surpass ± 25% with the ratio relative deviation of standard finger-print respective peaks peak area; Unimodal area accounts for total peak area than greater than 5% less than 10% peak in the total peak, must not surpass ± 30% with the ratio relative deviation of standard finger-print respective peaks peak area.

The thin-layer chromatography discrimination method of scutellarin in embodiment 5 injection sterile powders:

Get this product 0.05g, add methyl alcohol 20ml and extract, the preparation need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg; The test of employing thin-layered chromatography, draw each 3 μ l of above-mentioned solution, putting on same polyamide membrane with strip tape respectively, is developping agent with methyl alcohol-ethyl acetate-formic acid=7: 2: 1, launches, take out, dry, spray is with 2% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the streak of same color.

The thin-layer chromatography discrimination method of scutellarin in embodiment 6 injection sterile powders:

Get this product 0.1g, add 50% ethanol 20ml and extract, the preparation need testing solution; Other gets the scutellarin reference substance, adds 50% ethanol and makes the solution that every 1ml contains 1mg; The test of employing thin-layered chromatography, drawing each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, is developping agent with ethanol-ethyl formate-formic acid=8: 4: 1, launch, take out, dry, spray is with 5% aluminium choride ethanolic solution, 105 ℃ were dried by the fire 5 minutes, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

The thin-layer chromatography discrimination method of scutellarin in embodiment 7 parenteral solutions:

Get this product 2ml, evaporate to dryness, residue add methyl alcohol 10ml and extract, the preparation need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg; The test of employing thin-layered chromatography, draw each 2 μ l of above-mentioned solution, putting on same polyamide membrane with strip tape respectively, is developping agent with ethanol-butyl acetate-formic acid=5: 0.5: 3, launches, take out, dry, spray is with 4% ferric trichloride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the streak of same color.

The thin-layer chromatography discrimination method of scutellarin in the embodiment 8 injection dopes:

Get this product 1ml, with 5 times of amount ethanol dilutions, preparation need testing solution; Other gets the scutellarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg; The test of employing thin-layered chromatography, draw each 10 μ l of above-mentioned solution, putting respectively on same polyamide membrane, is developping agent with methyl alcohol-butyl acetate-glacial acetic acid=12: 5: 0.02, launches, take out, dry, spray is with 5% aluminium choride ethanolic solution, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

The liquid chromatography discrimination method of scutellarin in embodiment 9 injection sterile powders:

Sample thief 20mg adds methyl alcohol 20ml and extracts, and uses filtering with microporous membrane, gets subsequent filtrate as need testing solution; Get the scutellarin reference substance, add methyl alcohol and make the contrast solution that every 1ml contains 0.1mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-0.1% phosphate aqueous solution=50: 50 is a moving phase, and the detection wavelength is 335nm; Draw need testing solution and reference substance solution 10 μ l respectively, inject liquid chromatograph, test, in the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;

The liquid chromatography discrimination method of scutellarin in embodiment 10 concentrated solution for injection:

Sample thief 0.5ml adds ethanol 20ml dilution, the preparation need testing solution; Get the scutellarin reference substance, add methyl alcohol and make the contrast solution that every 1ml contains 0.05mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-3% glacial acetic acid aqueous solution=70: 30 is a moving phase, and the detection wavelength is 200nm; Draw need testing solution and reference substance solution 20 μ l respectively, inject liquid chromatograph, test, in the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;

The liquid chromatography discrimination method of scutellarin in embodiment 11 parenteral solutions:

Sample thief 2ml adds ethanol 20ml dilution, the preparation need testing solution; Get the scutellarin reference substance, add ethanol and make the contrast solution that every 1ml contains 0.05mg, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, acetonitrile-0.005mol/L potassium dihydrogen phosphate=16: 84 is a moving phase, and the detection wavelength is 410nm; Draw need testing solution and reference substance solution 5 μ l respectively, inject liquid chromatograph, test, in the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;

The thin-layer chromatography discrimination method of Paeoniflorin in embodiment 12 injection sterile powders:

Sample thief 0.2g adds methyl alcohol 20ml and extracts, the preparation need testing solution; Other gets the Paeoniflorin reference substance, adds methyl alcohol and makes the reference substance solution that every ml contains Paeoniflorin 1mg; The test of employing thin-layered chromatography, draw test solution and contrast solution 1～5 μ l, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methyl alcohol-formic acid=6: 1.5: 1.5: 0.5 was developping agent, launches, take out, dry, spray is with 5% ferric trichloride ethanolic solution, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the same color spot;

The thin-layer chromatography discrimination method of Paeoniflorin in embodiment 13 concentrated solution for injection:

Sample thief 0.5ml adds methyl alcohol 20ml dilution, the preparation need testing solution; Other gets the Paeoniflorin reference substance, adds methyl alcohol and makes the reference substance solution that every ml contains Paeoniflorin 2mg; The test of employing thin-layered chromatography, draw test solution and contrast solution 3 μ l, put respectively on same silica gel g thin-layer plate, with methylene chloride-ethyl acetate-methyl alcohol-formic acid=8: 0.5: 5: 4 was developping agent, launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, the heating dot colour developing is clear, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the same color spot;

The thin-layer chromatography discrimination method of Paeoniflorin in embodiment 14 parenteral solutions:

Sample thief 2ml adds ethanol 20ml dilution, the preparation need testing solution; Other gets the Paeoniflorin reference substance, adds ethanol and makes the reference substance solution that every ml contains Paeoniflorin 1mg; The test of employing thin-layered chromatography, it is an amount of to draw test solution and contrast solution, puts respectively on same silica gel g thin-layer plate, and with methylene chloride-butyl acetate-methyl alcohol-glacial acetic acid=2: 5: 5: 0.01 was developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, the heating dot colour developing is clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the same color spot;

The liquid chromatography discrimination method of Paeoniflorin in embodiment 15 injection sterile powders:

Sample thief 5mg adds methyl alcohol 20ml and extracts, and uses filtering with microporous membrane, gets subsequent filtrate as need testing solution; Get the Paeoniflorin reference substance and add methyl alcohol and make the reference substance solution that every ml contains 0.1mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-0.1% formic acid solution=40: 60 is a moving phase, and the detection wavelength is 230nm; Draw need testing solution and reference substance solution 5 μ l respectively, inject liquid chromatograph, test, in the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.

The liquid chromatography discrimination method of Paeoniflorin in embodiment 16 concentrated solution for injection:

Sample thief 0.2ml adds methyl alcohol 30ml dilution, the preparation need testing solution; Get the Paeoniflorin reference substance and add methyl alcohol and make the reference substance solution that every ml contains 0.1mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-0.001% phosphoric acid=15: 85 is moving phase, and the detection wavelength is 200nm; Draw need testing solution and reference substance solution 20 μ l respectively, inject liquid chromatograph, test, in the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.

The liquid chromatography discrimination method of Paeoniflorin in embodiment 17 parenteral solutions:

Sample thief 1ml adds ethanol 20ml dilution, the preparation need testing solution; Getting the Paeoniflorin reference substance adds 95% ethanol and makes the reference substance solution that every ml contains 0.05mg, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, and methyl alcohol-0.3mol/L potassium dihydrogen phosphate=62: 38 is moving phase, and the detection wavelength is 410nm; Draw need testing solution and reference substance solution 1～20 μ l respectively, inject liquid chromatograph, test, in the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.

The assay of scutellarin in embodiment 18 injection sterile powders:

Sample thief 20mg, the accurate title, decide, and adds methyl alcohol 50ml and extract, the preparation need testing solution; Get the scutellarin reference substance, add methyl alcohol and make the reference substance solution that every ml contains 0.1mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-0.1% phosphate aqueous solution=50: 50 is a moving phase, and the detection wavelength is 335nm; Respectively accurate need testing solution and the reference substance solution 10 μ l of drawing inject liquid chromatograph, test, and calculate with one point external standard method, and must not be less than 12mg with what dosage contained scutellarin every day in the preparation;

The assay of scutellarin in embodiment 19 concentrated solution for injection:

Precision is measured sample 0.2ml, adds methyl alcohol alcohol 50ml dilution, uses filtering with microporous membrane, gets subsequent filtrate as need testing solution; Get the scutellarin reference substance, add methyl alcohol and make the reference substance solution that every ml contains 0.05mg, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, methyl alcohol-0.005mol/L sodium dihydrogen phosphate=70: 30 is moving phase, and the detection wavelength is 200nm; Respectively accurate need testing solution and the reference substance solution 15 μ l of drawing inject liquid chromatograph, test, and calculate with one point external standard method, and must not be less than 20mg with what dosage contained scutellarin every day in the preparation;

The assay of scutellarin in embodiment 20 parenteral solutions:

Precision is measured sample 0.5ml, adds ethanol 50ml dilution, uses filtering with microporous membrane, gets subsequent filtrate as need testing solution; Get the scutellarin reference substance, add ethanol and make the reference substance solution that every ml contains 0.05mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-3% aqueous formic acid=16: 84 is a moving phase, and the detection wavelength is 410nm; Respectively accurate need testing solution and the reference substance solution 10 μ l of drawing inject liquid chromatograph, test, and calculate with calibration curve method, and must not be less than 25mg with what dosage contained scutellarin every day in the preparation;

Content of paeoniflorin is measured in embodiment 21 injection sterile powders:

Sample thief 20mg, the accurate title, decide, and adds methyl alcohol 50ml and extract, and uses filtering with microporous membrane, gets subsequent filtrate as need testing solution; Get the Paeoniflorin reference substance, add methyl alcohol and make the reference substance solution that every ml contains 0.1mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methyl alcohol-0.1% formic acid solution=40: 60 is a moving phase, and the detection wavelength is 230nm; Respectively accurate need testing solution and the reference substance solution 10 μ l of drawing inject liquid chromatograph, test, and calculate with one point external standard method, and must not be less than 30mg with what dosage contained Paeoniflorin every day in the preparation;

Content of paeoniflorin is measured in embodiment 22 injection sterile powders:

Sample thief 100mg, the accurate title, decide, and adds methyl alcohol 100ml and extract, and uses filtering with microporous membrane, gets subsequent filtrate as need testing solution; Get the Paeoniflorin reference substance, add methyl alcohol and make the reference substance solution that every ml contains 0.02mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-0.05% glacial acetic acid solution=15: 85 is a moving phase, and the detection wavelength is 200nm; Respectively accurate need testing solution and the reference substance solution 15 μ l of drawing inject liquid chromatograph, test, and calculate with calibration curve method, and must not be less than 15mg with what dosage contained Paeoniflorin every day in the preparation;

Content of paeoniflorin is measured in embodiment 23 parenteral solutions:

Precision is measured sample 1ml, adds methyl alcohol and is diluted to 10ml, the preparation need testing solution; Get the Paeoniflorin reference substance, add methyl alcohol and make the reference substance solution that every ml contains 0.05mg, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, methyl alcohol-0.3mol/L sodium dihydrogen phosphate=62: 38 is moving phase, and the detection wavelength is 410nm; Respectively accurate need testing solution and the reference substance solution 5 μ l of drawing inject liquid chromatograph, test, and calculate with calibration curve method, and must not be less than 20mg with what dosage contained Paeoniflorin every day in the preparation;

Content of paeoniflorin is measured in embodiment 24 concentrated solution for injection:

Precision is measured sample 0.2ml, adds methyl alcohol and is diluted to 25ml, uses filtering with microporous membrane, gets subsequent filtrate as need testing solution; Get the Paeoniflorin reference substance, add methyl alcohol and make the reference substance solution that every ml contains 0.05mg, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, and acetonitrile-3% aqueous formic acid=30: 70 is a moving phase, and the detection wavelength is 330nm; Respectively accurate need testing solution and the reference substance solution 20 μ l of drawing inject liquid chromatograph, test, and calculate with calibration curve method, and must not be less than 30mg with what dosage contained Paeoniflorin every day in the preparation;

Claims

1. the method for quality control of a pharmaceutical composition made from Breviscapinun, Paeoniflorin, 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 Breviscapinun, Paeoniflorin of claim 1, it is characterized in that this method comprises all or part of content of following finger-print test:

II. non-total peak area must not surpass 10% of total peak area;

3. according to the method for quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony of claim 2, it is characterized in that this method comprises all or part of content of following finger-print test:

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Breviscapinun, the root of herbaceous peony or radix paeoniae rubrathe composition characteristics; Accurate respectively object of reference solution and each 10 μ 1 of need testing solution of drawing, inject liquid chromatograph 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 2～6;

II. non-total peak area must not surpass 5% of total peak area;

4. according to the method for quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony of claim 1, it is characterized in that this method comprises all or part of content in the following differential test method:

5. according to the method for quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony of claim 4, it is characterized in that this method comprises all or part of content in the following differential test method:

6. according to the method for quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony of claim 1, it is characterized in that this method comprises all or part of content of following assay:

The assay of scutellarin in a, the pharmaceutical composition:

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

7. according to the method for quality control of the described pharmaceutical composition made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony of claim 6, it is characterized in that this method comprises all or part of content of following assay:

The assay of scutellarin in a, the pharmaceutical composition:

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

8. according to the method for quality control of claim 6 or the 7 described pharmaceutical compositions made from Breviscapinun, the radix paeoniae rubrathe or the root of herbaceous peony, it is characterized in that: the assay result of described pharmaceutical composition, calculate according to percentage by weight, the total amount of scutellarin and Paeoniflorin accounts for deduction auxiliary material and outer more than 50% of total solid of moisture in the preparation.