CN1951417A - Quality control method of a pharmaceutical composition - Google Patents

Quality control method of a pharmaceutical composition Download PDF

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Publication number
CN1951417A
CN1951417A CNA2005101093964A CN200510109396A CN1951417A CN 1951417 A CN1951417 A CN 1951417A CN A2005101093964 A CNA2005101093964 A CN A2005101093964A CN 200510109396 A CN200510109396 A CN 200510109396A CN 1951417 A CN1951417 A CN 1951417A
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China
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solution
methanol
pharmaceutical composition
water
ginsenoside
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CNA2005101093964A
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于文风
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Qiyuanyide Medicines Institute Beijing
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Qiyuanyide Medicines Institute Beijing
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Priority to CNA2005101093964A priority Critical patent/CN1951417A/en
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Abstract

The invention relates to a method of quality control for medicinal compositions prepared from Breviscapine, pseudo-ginseng, astragalus root or the extract and significant portions of them, which comprises a fingerprint pattern testing method mainly featuring the constituent characteristic of astragalus root flavones, astragalus root saponins and pseudo-ginseng root saponins, and/or differentiation testing method and/or content determination method for Breviscapine, pseudo-ginseng and astragalus root. The invention provides a reliable, stable and novel method for quality control of the preparation, which realizes more stricter and reasonable process control, more stable quality, more reliable detection method for mass production, and digitalized illustration for guaranteeing safety and effectiveness of patients' drug administration is provided.

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 medicaments.
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, oneself becomes commonly encountered diseases, the frequently-occurring disease of harm China people ' s health ischemic cardiovascular and cerebral vascular disease; 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 scutellarin, astragaloside, Panax Notoginseng saponin R 1, the ginsenoside Rg 1With certain composition among the ginsenoside Rb1 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):
Be made for 10～5000 parts by 0.1～50 part of breviscapine, 10～5000 parts of the Radixs Astragali and Radix Notoginseng; Or add suitable adjuvant and be made through extracting the extract that obtains after refining and corresponding weight portion breviscapine by corresponding weight portion Milkvetch Root, pseudo-ginseng.
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 pharmaceutical composition is characterized in that: this method comprises following all or part of content:
(1) finger printing test comprises with the Radix Astragali flavone constituents being characterized as main finger printing and being characterized as in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents;
(2) Radix Astragali control medicinal material, Radix Notoginseng control medicinal material, scutellarin, astragaloside, formononetin, calycosin, Panax Notoginseng saponin R 1, the ginsenoside Rg 1With ginsenoside Rb 1In the differential test method of all or part of composition;
(3) scutellarin, astragaloside, formononetin, calycosin, Panax Notoginseng saponin R 1, the ginsenoside Rg 1, ginsenoside Rb 1, the content test method of all or part of composition in the total saponins.
The method of quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali is characterized in that: this method adopts liquid chromatography test to be characterized as main finger printing and to be characterized as in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents:
A, the test of employing liquid chromatography are characterized as main finger printing with the Radix Astragali flavone constituents:
(1) preparation of need testing solution: the 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 formononetin,, be settled to suitable concn, as object of reference solution with methanol or dissolve with ethanol;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol-water or 0.1%～5% glacial acetic acid or 0.1%～5% formic acid or 0.005%～5% phosphoric acid solution, gradient elution, flow velocity is that 0.5～2.0ml/min, detection wavelength are one or several in the 190-400nm scope, and column temperature is in 20～60 ℃ of scopes;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～50;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the pharmaceutical composition to be measured with the Radix Astragali flavone constituents, the preparation testing sample finger printing;
(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 relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 20%;
B, the test of employing liquid chromatography are characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents:
(1) preparation of need testing solution: it is an amount of to get pharmaceutical composition to be measured, adds methanol or ethanol or water and is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Astragali and the pseudo-ginseng, comprise the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1, a kind of in the astragaloside, water or methanol or dissolve with ethanol are settled to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol-water or 0.1%～5% glacial acetic acid or 0.1%～5% formic acid or 0.005%～5% phosphoric acid solution, gradient elution, flow velocity is 0.5～2.0ml/min, detect wavelength is that one or several or evaporative light scattering detector in 190～400nm scope detects, and column temperature is in 20～60 ℃ of scopes;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～40;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the pharmaceutical composition to be measured with Radix Astragali saponin class, arasaponin constituents, the preparation testing sample finger printing;
(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 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%.
The method of quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali is characterized in that: this method adopts liquid chromatography test to be characterized as main finger printing and to be characterized as in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents:
A, the test of employing liquid chromatography are characterized as main finger printing with the Radix Astragali flavone constituents:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing pharmaceutical composition to be measured, adds water and make the solution that every 1ml contains 1mg, shakes up, promptly;
(2) preparation of object of reference solution: get formononetin, with dissolve with methanol and be diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-water, gradient elution, solvent ratios is from 0 minute to 20 minutes, the ratio of acetonitrile is for to rise to 25% from 4%, from 20 minutes to 40 minutes, the ratio of acetonitrile is for to rise to 45% from 25%, from 40 minutes to 60 minutes, the ratio of acetonitrile is for rising to 70% from 45%, and from 60 minutes to 70 minutes, the ratio of acetonitrile was for to rise to 80% from 70%, from 70 minutes to 80 minutes, the ratio of acetonitrile is for rising to 100% from 80%, and flow velocity is 1ml/min, the detection wavelength is 254 ± 2nm, 25 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the pharmaceutical composition to be measured with the Radix Astragali flavone constituents, the preparation testing sample finger printing;
(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 0.90～1.00;
II. in the pharmaceutical composition finger printing to be measured, non-total peak area must not surpass 5% of total peak area;
III. the 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%;
B, the test of employing liquid chromatography are characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing pharmaceutical composition to be measured, adds methanol and make the solution that every 1ml contains 50mg, shakes up, promptly;
(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg 1In right amount, add methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-water, gradient elution, solvent ratios is from 0 minute to 14 minutes, the ratio of acetonitrile is 15%, and from 14 minutes to 45 minutes, the ratio of acetonitrile was for to rise to 30% from 15%, from 45 minutes to 60 minutes, the ratio of acetonitrile is for rising to 80% from 30%, and flow velocity is 1ml/min, detect wavelength 203 ± 2nm, 30 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～20;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the pharmaceutical composition to be measured with Radix Astragali saponin class, arasaponin constituents, the preparation testing sample finger printing;
(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 0.90～1.00;
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%.
The method of quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali is characterized in that: the discrimination method of described pharmaceutical composition comprises one or more in following:
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 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 scutellarin reference substance, adds methanol or ethanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or polyamide film, with ethyl acetate or Ethyl formate-formic acid or acetic acid-water or methanol 0.2～60: 0.2～10: 0.3～20 or benzene or toluene-ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1～30: 0.5～15: 0.05～5: 0.01～3 is developing solvent, launch, take out, dry, spray is with 0.3%～10% aluminum chloride ethanol or 0.3～10% aluminum chloride methanol solution or 0.3～10% ferric chloride alcoholic solution, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect or 1～15 minute rearmounted daylight of 105 ℃ of bakings is inspected down or under ultra-violet lamp 365nm or the 254nm, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of scutellarin in b, 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 scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, with methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution 5%～95%: 95%～5% or methanol or acetonitrile-oxolane-0.01%～5% phosphate aqueous solution 2%～30%: 2%～30%: 96%～40% or acetonitrile-0.005 moL/L～0.3moL/L sodium dihydrogen phosphate (1～99% phosphoric acid is transferred pH=2.0～5.0) gradient elution system is mobile phase, and the detection wavelength is 200～410nm; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
Radix Notoginseng, ginsenoside Rg in c, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more thin layer chromatography discrimination method:
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 Notoginseng control medicinal material, ginsenoside Rg 1Reference substance, ginsenoside Rb 1Reference substance, Panax Notoginseng saponin R 1In the reference substance one or more, the preparation contrast solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adding chloroform or dichloromethane or aether backflow extracts, discard chloroform or dichloromethane or ether solution, residue adds water-saturated n-butanol or ethyl acetate extraction, extracting solution adds ammonia solution, divides and gets upper strata, evaporate to dryness, residue is with methanol or dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more reference substances, add methanol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, lower floor's solution or n-butyl alcohol-ethyl acetate or the Ethyl formate-water 1～10: 0.2～2 placed below 5～40: 10～100: 5～50: 0.2～30 10 ℃ with chloroform-ethyl acetate or Ethyl formate-methanol-water: 1～15 upper strata is developing solvent, launch, take out, dry, spray is with 5～50% sulphuric acid ethanol reagent or 50% sulphate reagent, 80 ℃～160 ℃ dry by the fire to speckle colour developing clear, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color;
Ginsenoside Rg in d, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more liquid chromatograph differentiate:
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, Panax Notoginseng saponin R 1In the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution 5%～40%: 95%～60% are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
The thin layer chromatography discrimination method of the Radix Astragali in e, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, add ethyl acetate or ethanol or methanol or n-butanol extraction, filter the filtrate evaporate to dryness, residue adds the dissolving of 0.05%～5% sodium hydroxide solution, filter, filtrate is with hydrochloric acid condition pH value to 3～6, with ethyl acetate or n-butanol extraction, filter, filtrate evaporate to dryness, residue add ethyl acetate or methanol or dissolve with ethanol, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or polyamide film, with chloroform or methylene chloride-methanol or ethanol 1～30: 0.2～10 is developing solvent, launch, take out, dry, put to smoke under the rearmounted daylight or under ultra-violet lamp 365nm or the 254nm in the ammonia steam and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;
The thin layer chromatography discrimination method of astragaloside in f, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, be added on the neutral alumina post after adding methanol or dissolve with ethanol,, collect eluent with 10%～70% methanol-eluted fractions, evaporate to dryness, add water-saturated n-butanol after residue is dissolved in water and extract, merge n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or polyamide film, lower floor's solution with chloroform or methylene chloride-methanol or alcohol-water 1～30: 1～20: 0.2～10 is developing solvent, launch, take out, dry, spray is with 2%～50% ethanol solution of sulfuric acid, 80～160 ℃ to be heated to speckle colour developing clear, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of astragaloside in g, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, and it is back with n-butyl alcohol or ethyl acetate extraction, merge extractive liquid, to be dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol or 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 15%～85%: 85%～15% methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, detecting wavelength is that 190～410nm or evaporative light scattering detector detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
The thin layer chromatography discrimination method of one or both of formononetin in h, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Other gets in formononetin, the hair stamen formononetin reference substance one or both, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, with chloroform or methylene chloride-methanol or ethanol-ethyl acetate or Ethyl formate-water 0.5～10: 1～15: 1～20: 0.1～3 is developing solvent, launch, take out, dry, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect, in the test sample chromatograph, with reference substance chromatograph and the corresponding position of control medicinal material on, should show the same color speckle;
The liquid chromatograph discrimination method of one or both of formononetin in i, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Methanol or alcoholic solution with formononetin, hair stamen formononetin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, mobile phase A is methanol or acetonitrile, Mobile phase B is that water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, gradient elution, the detection wavelength is 190～410nm, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
The method of quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali is characterized in that: the discrimination method of described pharmaceutical composition comprises one or more in following:
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 methanol extraction, centrifugal, gets supernatant as need testing solution; Other gets the scutellarin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, put on same polyamide membrane with strip tape respectively, be developing solvent with methanol-ethyl acetate-formic acid at 7: 2: 1, launches, take out, dry, spray is with 2% ferric chloride alcoholic solution, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the streak of same color;
The liquid chromatograph discrimination method of scutellarin in b, 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 or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution is a mobile phase at 50%: 50%, and the detection wavelength is 335nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
Radix Notoginseng, ginsenoside Rg in c, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more thin layer chromatography discrimination method:
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 Notoginseng control medicinal material, ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more, the preparation contrast solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adds the chloroform reflux, extract,, discards chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances in ginsenoside Rg1, ginsenoside Rbl, the arasaponin R1, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: 10 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;
Ginsenoside Rg in d, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more liquid chromatograph differentiate:
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, Panax Notoginseng saponin R 1In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
The thin layer chromatography discrimination method of the Radix Astragali in e, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, adds ethanol extraction, filters the filtrate evaporate to dryness, residue adds the dissolving of 0.3% sodium hydroxide solution, filters, and filtrate is used ethyl acetate extraction with hydrochloric acid condition pH value to 5～6, filter, filtrate evaporate to dryness, residue add the ethyl acetate dissolving, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developing solvent with chloroform-methanol at 10: 1, launches, take out, dry, put in the ammonia steam and inspect under the smoked rearmounted ultra-violet lamp 365nm, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle;
The thin layer chromatography discrimination method of astragaloside in f, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, be added on the neutral alumina post after adding dissolve with methanol, use 40% methanol-eluted fractions, collect eluent, evaporate to dryness, add water-saturated n-butanol after residue is dissolved in water and extract, merge n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of 13: 7: 2 of chloroform-methanol-water is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts to reach under the ultra-violet lamp 365nm daylight under and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of astragaloside in g, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, and back water saturation n-butanol extraction, merge extractive liquid, are dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol or 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
The thin layer chromatography discrimination method of one or both of formononetin in h, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, and extracting solution is as need testing solution; Other gets in formononetin, the hair stamen formononetin one or both, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-ethyl acetate-water 2.5: 3: 4: 0.5 was developing solvent, launch, exhibition is taken out apart from 5cm, dries, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle;
The liquid chromatograph discrimination method of one or both of formononetin in i, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, and extracting solution is as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
The method of quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali is characterized in that: the method for testing of described pharmaceutical composition content should comprise one or more in following:
The assay of scutellarin in a, 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 scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, with methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution 5%～95%: 95%～5% or or methanol or acetonitrile-oxolane-0.01%～5% phosphate aqueous solution 2%～30%: 2%～30%: 96%～40% or acetonitrile-0.005moL/L～0.3moL/L sodium dihydrogen phosphate (1～99% phosphoric acid is transferred pH=2.0～5.0) gradient elution system be mobile phase, the detection wavelength is 200～410nm; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 2mg;
Ginsenoside Rg in b, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more assay:
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, Panax Notoginseng saponin R 1In the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with external standard method or standard curve method, 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 per unit amount contains the ginsenoside Rg 1Limit must not be less than 2.5mg;
The per unit amount contains ginsenoside Rb 1Limit must not be less than 3.5mg;
The per unit amount contains Panax Notoginseng saponin R 1Limit must not be less than 0.25mg;
The per unit amount contains the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The limit of summation must not be less than 6mg;
The assay of total saponins in c, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, put in the measuring bottle, adding distil water makes dissolving in right amount and decides and shakes up to scale, and precision is measured in right amount, put in the measuring bottle, water bath method takes out immediately, and precision adds 1%～50% vanillin-glacial acetic acid solution 0.1～10ml, perchloric acid 0.1～15ml, shake up, heated 3～50 minutes in 30～80 ℃ of water-baths, take out, immediately with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale, shakes up, as need testing solution, with astragaloside or ginsenoside Rg1 or ginsenoside Rb1 or arasaponin R1 is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures trap, calculate with one point external standard method or standard curve method, 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 astragaloside or ginsenoside Rg 1Or ginsenoside Rb 1Or Panax Notoginseng saponin R 1Meter must not be less than 10mg;
The assay of astragaloside in d, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, and it is back with n-butyl alcohol or ethyl acetate extraction, merge extractive liquid, to be dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 15%～85%: 85%～15% methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, detecting wavelength is that 190～410nm or evaporative light scattering detector detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.02mg;
The assay of one or both of formononetin in e, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Methanol or alcoholic solution with formononetin, hair stamen formononetin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, mobile phase A is methanol or acetonitrile, Mobile phase B is that water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, gradient elution, the detection wavelength is 190～410nm, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, 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 two:
(1) the per unit amount limit that contains formononetin must not be less than 0.01mg;
(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.01mg.
The method of quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali, it is characterized in that: the method for testing of described pharmaceutical composition content is following one or more methods:
The assay of scutellarin in a, 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 or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution 50%:50% is a mobile phase, and the detection wavelength is 335nm; A bit calculate with external standard, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg;
Ginsenoside Rg in b, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more assay:
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, Panax Notoginseng saponin R 1In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with external standard method or standard curve method, 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 5mg;
(2) the per unit amount contains ginsenoside Rb 1Limit must not be less than 7mg;
(3) the per unit amount contains Panax Notoginseng saponin R 1Limit must not be less than 0.5mg;
(4) the per unit amount contains the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The limit of summation must not be less than 12mg;
The assay of total saponins in c, the pharmaceutical composition:
It is an amount of to get medicine to be measured, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision measures 0.6, puts in the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin, one glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with the ginsenoside Rg 1Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, 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 20mg;
The assay of astragaloside in d, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, and back water saturation n-butanol extraction, merge extractive liquid, are dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.04mg;
The assay of one or both of formononetin in e, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, and extracting solution is as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 30 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, 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 two:
(1) the per unit amount limit that contains formononetin must not be less than 0.02mg;
(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.02mg.
The method of quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali, it is characterized in that: the assay result of the ejection preparation of described compositions, calculate scutellarin, astragaloside, formononetin, calycosin, ginsenoside Rg according to percentage by weight 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1, all or part of material in the total saponins total content account for deduction adjuvant and outer more than 25% of total solid of moisture in the preparation.
Compared with prior art, the present invention's quality of the pharmaceutical composition made with breviscapine, Radix Notoginseng, the Radix Astragali of perfect control more.The Chinese medicine ingredients complexity, if only with wherein one, two kind of composition illustrate its inherent quality, has certain one-sidedness, more can't judge the index components of its drug effect.Therefore the applicant has formulated with the Radix Astragali flavone constituents and has been characterized as main finger printing and is characterized as the quality that main finger printing, discriminating and assay are controlled pharmaceutical composition comprehensively with Radix Astragali saponin class, arasaponin constituents.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 in the pharmaceutical composition being characterized as main finger printing and being characterized as main finger printing characteristic peak changing with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents, and have only the condition of the present invention of employing, just can obtain ideal thin layer chromatography, contain survey condition and finger printing.
Proof by experiment, method of quality control of the present invention is more effective to the quality control of the pharmaceutical composition product made with breviscapine, Radix Notoginseng, the Radix Astragali, and method precision, stability are all higher.
Experimental example 1 is characterized as main finger printing with the Radix Astragali flavone constituents
A, experimental apparatus, reagent and sample:
Reference substance: formononetin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
B, chromatographic condition and system suitability experiment:
1. the selection of chromatographic column:
In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, has tried out Inertsil ODS-3 (250mm * 4.6mm, 5 μ m) Kromasil C respectively 18(200mm * 4.6mm, 5 μ m), Diamonsil C 18The chromatographic column of (250mm * 4.6mm, 5 μ m) three kinds of trades mark, the result shows that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, wherein Diamonsil C 18The chromatographic column separating effect is best, and post is imitated the highest (calculating with the object of reference formononetin).So finally select Diamonsil C for use 18(250mm * 4.6mm, 5 μ m) are the experimentation post.
2. the selection of mobile phase:
Investigated (1) methanol-0.05mol/L sodium dihydrogen phosphate (20: 80) in the research process respectively, (2) acetonitrile-0.05mol/L sodium dihydrogen phosphate (10: 90), (3) acetonitrile-0.05mol/L sodium hydrogen phosphate (gradient elution) (4) acetonitrile-water (gradient elution), solvent ratios is from 0 minute to 20 minutes, the ratio of acetonitrile is for to rise to 25% from 4%, from 20 minutes to 40 minutes, the ratio of acetonitrile is for to rise to 45% from 25%, from 40 minutes to 60 minutes, the ratio of acetonitrile is for rising to 70% from 45%, and from 60 minutes to 70 minutes, the ratio of acetonitrile was for to rise to 80% from 70%, from 70 minutes to 80 minutes, the ratio of acetonitrile was for to rise to 100% from 80%.The result shows that peak shape is relatively poor under mobile phase (1) condition, and it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (2) the mobile phase condition, peak shape is relatively poor, separates bad; (3) under the condition, the peak hangover is serious, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.
3. detection wavelength determination:
In the research under acetonitrile-water (gradient elution) mobile phase condition, investigated the chromatographic peak situation under different-waveband typical wavelengths 230,254,270,300 respectively, the result shows, 254nm can take into account kurtosis, separating degree and the baseline of each composition chromatographic peak when detecting, so select 254nm to detect wavelength for this product finger printing.
4. instrument, chromatographic column and integral parameter:
4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved LC-10Avp high performance liquid chromatograph for use, the WML-2010 chromatographic work station.Chromatographic column is Diamonsil C 18(250mm * 4.6mm, 5 μ m); 40 ℃ of column temperatures, flow velocity 1.0ml/min.
4.2 integral parameter: peak width: 10; Slope: 100; Smallest peaks area: 500.So setting can be avoided some very calculating of the chromatographic peak of small size (unimodal area accounts for total peak area less than 0.5%), guarantees the dependency with object of reference simultaneously.
5. the preparation of need testing solution:
Get pharmaceutical composition 0.1g to be measured, add the n-butyl alcohol dissolving, filter, filtrate evaporate to dryness, residue add methanol and are diluted to 5ml, as need testing solution.
6. the preparation of object of reference solution:
Get formononetin, add methanol and make the solution that every 1ml contains 0.1mg, as object of reference solution.
7. finger printing and technical parameter:
Formulate standard finger-print according to 10 batch samples, test sample finger printing and standard finger-print are compared, similarity is all between 0.90～1.00.
Experimental example 2 is characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents
A, experimental apparatus, reagent and sample:
Reference substance: ginsenoside Rg 1: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
B, chromatographic condition and system suitability experiment:
1. the selection of chromatographic column:
In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, tried out Zorbax, Inertsil ODS-3 respectively, Diamonsil ODS (is C18,4.6mm * 200mm, 5 μ m) chromatographic column of three kinds of trades mark, result show that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, and wherein Diamonsil ODS chromatographic column separating effect is best, post is imitated the highest, can reach 5400 (with the object of reference ginsenoside Rg 1Calculate).So finally selecting Diamonsil ODS chromatographic column (4.6mm * 200mm, 5 μ m) for use is the experimentation post.
2. the selection of mobile phase:
Investigated (1) methanol-water (20: 80) in the research process respectively, (2) acetonitrile-water (10: 90), (3) (the gradient elution volume proportion is from 0 minute to 14 minutes to acetonitrile-0.05mol/L sodium dihydrogen phosphate (gradient elution) (4) acetonitrile-water (gradient elution), the ratio of acetonitrile is 15%, from 14 minutes to 45 minutes, the ratio of acetonitrile is for rising to 30% from 15%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was for rising to 80% from 30%) four kinds of flow phase system.The result shows that peak shape is relatively poor under mobile phase (1) condition, and it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (2) the mobile phase condition, peak shape is relatively poor, separates bad; (3) under the condition, the peak hangover is serious, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.
3. detection wavelength determination:
In the research under acetonitrile-water (gradient elution) mobile phase condition, investigated the chromatographic peak situation under different-waveband typical wavelengths 203,210,230,254 respectively, the result shows that chromatographic peak is more under 203nm, peak shape is better, so finally select for use 203nm ± 2 as detecting wavelength.
4. instrument, chromatographic column and integral parameter:
4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved Agilent1100 series of high efficiency chromatograph of liquid for use, the Chemstation chromatographic work station.Chromatographic column is Diamonsil ODS (4.6mm * 200mm, 5 μ m); 40 ℃ of column temperatures, flow velocity 1.0ml/min.
4.2 integral parameter: Slope Sensitivity:1, peak width:0.05, smallest peaks area are 5% of object of reference (S) peak-to-peak area, minimum peak height be the S peak-to-peak high 5%.So setting can be avoided some very calculating of the chromatographic peak of small size (unimodal area accounts for total peak area less than 0.5%), guarantees the dependency with object of reference simultaneously.
5. the preparation of need testing solution:
It is an amount of that precision takes by weighing this product, adds water and make the solution that every 1ml contains 50mg, promptly.
6. the preparation of object of reference solution:
The ginsenoside Rg 1Be one of Radix Notoginseng main active, its integral area proportion in finger printing more greatly and more stable is taken into account the research of intermediate and medical material simultaneously, therefore selected ginsenoside Rg 1As object of reference.
7. finger printing and technical parameter:
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 scutellarin in experimental example 3 pharmaceutical compositions:
Feature for outstanding Herba Erigerontis, selected scutellarin as its feature, but owing to there is composition like more, the polar phase close in the medical material with the scutellarin structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition launches scutellarin:
Condition question
Normal hexane-benzene-Ethyl formate-formic acid (5-5-10-4) silica gel H lamellae reference substance is expanded to the forward position
Ethyl acetate-benzene-acetic acid (5-4-3) silica gel H lamellae reference substance does not separate, and feminine gender has interference
Ethyl acetate-benzene-formic acid (5-4-3) silica gel g thin-layer plate reference substance does not separate, and feminine gender has interference
Chloroform-ethyl acetate-methanol (7-2-4) silica GF254 lamellae reference substance does not separate, and feminine gender has interference
Toluene-ethyl acetate (8-1) silica gel H lamellae reference substance does not separate, and feminine gender has interference
Methanol-ethyl acetate-formic acid (10-5-0.5) silica gel g thin-layer plate reference substance is expanded to the forward position
It is clear that methanol-ethyl acetate-formic acid (7-2-1) polyamide membrane is separated, and the moderate feminine gender of Rf value is noiseless
Through screening, determined with the polyamide membrane to be immobile phase, be developing solvent with methanol-ethyl acetate-formic acid (7-2-1), with this understanding, the Rf value of scutellarin is moderate, and it is clear to separate with other speckle, negative noiseless.
The liquid chromatograph discrimination method of scutellarin in experimental example 4 pharmaceutical compositions:
Feature for outstanding Herba Erigerontis, except the thin layer discrimination method, selected scutellarin as its characteristic component, but owing to there is composition like more, the polar phase close in the medical material with the scutellarin structure, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase scutellarin are separated:
Condition Problem
Methanol-0.05mol/L sodium hydrogen phosphate (80: 20) eight alkyl silane bonded silica gels Appearance time is too fast
Acetonitrile-0.05mol/L sodium hydrogen phosphate (40: 60) octadecylsilane chemically bonded silica Feminine gender has interference
Methanol-oxolane-0.05mol/L sodium hydrogen phosphate (20: 10: 70) octadecylsilane chemically bonded silica Feminine gender has interference
Acetonitrile-oxolane-0.05mol/L sodium hydrogen phosphate (20: 10: 70) octadecylsilane chemically bonded silica Peak shape is slightly asymmetric
Methanol-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane chemically bonded silica Feminine gender has interference
Acetonitrile-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane chemically bonded silica Peak shape is slightly asymmetric
Methanol-0.1% phosphate aqueous solution (50: 50) octadecylsilane chemically bonded silica Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, methanol-0.1% phosphate aqueous solution (50: 50) is a mobile phase, and with this understanding, the scutellarin retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless.
Experimental example 5 pharmaceutical composition ginsenoside Rgs 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The thin layer chromatography discrimination method:
For the feature of outstanding Radix Notoginseng, selected the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1As its feature speckle, but owing to exist more and the ginsenoside Rg in the medical material 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1Structure is close, composition like the polar phase, and usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:
Condition Problem
Lower floor's solution silica gel g thin-layer plate that chloroform-Ethyl formate--water (20: 60: 10) is placed below 10 ℃ Reference substance is expanded to the forward position
N-butyl alcohol-Ethyl formate-methanol (10-1-5) silica gel H lamellae Reference substance is expanded to the forward position
Chloroform-Ethyl formate-formic acid-water (15: 40: 22: 10) lower floor's solution silica gel g thin-layer plate of placing below 10 ℃ Reference substance does not separate, and feminine gender has interference
Chloroform-ethyl acetate-formic acid-water (10: 40: 15: 5) lower floor's solution silica gel g thin-layer plate of placing below 10 ℃ Reference substance does not separate, and feminine gender has interference
N-butyl alcohol-Ethyl formate-methanol (5-1-5) silica gel g thin-layer plate Reference substance does not separate, and feminine gender has interference
N-butyl alcohol-Ethyl formate-ethanol (2-1.5-0.5) silica gel H lamellae Reference substance does not separate, and feminine gender has interference
Determine condition: chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower floor's solution silica gel g thin-layer plate of placing below 10 ℃ It is clear to separate, and the moderate feminine gender of Rf value is noiseless
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, with chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developing solvent, with this understanding, the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1Rf value moderate, it is clear to separate with other speckle, negative noiseless.
Ginsenoside Rg in experimental example 6 pharmaceutical compositions 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The liquid chromatograph discrimination method:
For the feature of outstanding Radix Notoginseng, selected the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1As its characteristic component, but owing to exist more and the ginsenoside Rg in the medical material 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1Structure is close, composition like the polar phase, and usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and mobile phase to the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1Separate:
Condition Problem
Methanol-0.05mol/L sodium dihydrogen phosphate (70: 30) octadecylsilane chemically bonded silica Appearance time is too fast
Acetonitrile-0.05mol/L sodium dihydrogen phosphate (55: 45) octadecylsilane chemically bonded silica Appearance time is too fast
Methanol-0.05mol/L sodium hydrogen phosphate (80: 20) eight alkyl silane bonded silica gels Feminine gender has interference
Methanol-0.05mol/L sodium hydrogen phosphate (80: 20) octadecylsilane chemically bonded silica Feminine gender has interference
Acetonitrile-0.02mol/L potassium dihydrogen phosphate (90: 10) eight alkyl silane bonded silica gels Feminine gender has interference
Methanol-0.02mol/L potassium dihydrogen phosphate (85: 15) octadecylsilane chemically bonded silica Feminine gender has interference
Acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute complete 15 minutes, and the ratio of acetonitrile was 20%, from 15 minutes to 21 minutes, the ratio of acetonitrile is moderate by retention time on 20%, the capable point in peak rises to 40%, and from 21 minutes to 25 minutes, the ratio of acetonitrile was that 20% octadecyl silicon is sharp, symmetry, negative noiseless
The alkane bonded silica gel
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile is 20%, with this understanding, and the people ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless.
The thin layer chromatography discrimination method of the Radix Astragali in experimental example 7 pharmaceutical compositions
Feature for the outstanding Radix Astragali, selected Radix Astragali control medicinal material as its feature speckle, but owing to there is composition like more, the polar phase close in the pharmaceutical composition with Radix Astragali control medicinal material structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition launches Radix Astragali control medicinal material:
Condition Problem
N-butanol-glacial acetic acid-water (6: 4: 3) silica gel H lamellae acetone-glacial acetic acid-water (8: 2: 1) silica gel g thin-layer plate methylene chloride-methanol (7-3) silica GF254 lamellae chloroform-acetone-formic acid (20: 2: 1) silica gel g thin-layer plate chloroform-acetone-methyl alcohol (20: 3: 2) silica gel H lamellae chloroform-acetone-methyl alcohol (20: 2: 10) silica gel g thin-layer plate chloroform-methyl alcohol (10-1) silica gel g thin-layer plate It is clear that feminine gender has the unintelligible feminine gender of interference characteristic spot to have the feminine gender of interference to have the feminine gender of interference to have the reference substance of interference to be expanded to the forward position separation, and the moderate feminine gender of Rf value is noiseless
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with chloroform-methanol (10-1), with this understanding, the Rf value of Radix Astragali control medicinal material feature speckle is moderate, and it is clear to separate with other speckle, negative noiseless.
The thin layer chromatography discrimination method of astragaloside in experimental example 8 pharmaceutical compositions
Feature for the outstanding Radix Astragali, selected astragaloside as its feature speckle, but owing to there is composition like more, the polar phase close in the pharmaceutical composition with the astragaloside structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition launches astragaloside:
Condition Problem
Chloroform-acetone-water (10-8-0.5) silica gel g thin-layer plate carrene-acetone-water (10-8-0.5) silica gel g thin-layer plate carrene-acetone (2-9) silica gel H lamellae chloroform-acetone (10-5) silica GF254 lamellae chloroform-ethanol (13-7) silica gel H lamellae chloroform-methyl alcohol (13-7) silica gel g thin-layer plate chloroform-methanol-water (13-7-2) lower floor solution Feminine gender has the feminine gender of interference to have the reference substance of interference exhibition to forward position feminine gender to have the feminine gender of interference to have the feminine gender of interference to have interference separation clear, and the moderate feminine gender of Rf value is noiseless
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with lower floor's solution of chloroform-methanol-water (13-7-2), with this understanding, the Rf value of astragaloside feature speckle is moderate, and it is clear to separate with other speckle, and feminine gender is noiseless.
The liquid chromatograph discrimination method of astragaloside in experimental example 9 pharmaceutical compositions
Feature for the outstanding Radix Astragali, except the thin layer discrimination method, selected astragaloside as its characteristic component, but owing to there is composition like more, the polar phase close in the pharmaceutical composition with the astragaloside structure, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase astragaloside are separated:
Condition Problem
Methanol-0.02mol/L sodium hydrogen phosphate (70: 30) eight alkyl silane bonded silica gels Appearance time is too fast
Acetonitrile-0.02mol/L sodium hydrogen phosphate (50: 50) octadecylsilane chemically bonded silica Appearance time is too fast
Methanol-0.05mol/L sodium hydrogen phosphate (40: 60) octadecylsilane chemically bonded silica Feminine gender has interference
Acetonitrile-0.05mol/L sodium hydrogen phosphate (30: 70) octadecylsilane chemically bonded silica Peak shape is slightly asymmetric
Methanol-oxolane-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane chemically bonded silica Feminine gender has interference
Acetonitrile-methanol-1% glacial acetic acid aqueous solution (20: 10: 70) octadecylsilane chemically bonded silica Peak shape is slightly asymmetric
Acetonitrile-water (32: 68) octadecylsilane chemically bonded silica Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water (32: 68) is a mobile phase, and with this understanding, the astragaloside retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless.
The thin layer chromatography discrimination method of formononetin, hair stamen formononetin in experimental example 10 pharmaceutical compositions
Feature for outstanding Radix Astragali flavone constituents, selected formononetin, hair stamen formononetin as its feature speckle, but because have in the pharmaceutical composition that more and formononetin, hair stamen formononetin structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition launches formononetin, hair stamen formononetin:
Condition Problem
Benzene-acetone-methyl alcohol (5-3-1) silica gel g thin-layer plate toluene-ethyl acetate-water (10-8-0.5) silica gel g thin-layer plate n-hexane-methyl alcohol (6-0.5) silica gel H lamellae cyclohexane-acetone (5-5) silica GF254 lamellae benzene-Ethyl formate-methyl alcohol (6-2-1) silica gel H lamellae toluene-n-hexane-methyl alcohol (8-4-4) silica gel g thin-layer plate chloroform-methyl alcohol-ethyl acetate-water (2.5-3-4-0.5) silica gel g thin-layer plate Feminine gender has the feminine gender of interference to have the feminine gender of interference to have the reference substance of interference exhibition to forward position feminine gender to have the feminine gender of interference to have interference separation clear, and the moderate feminine gender of Rf value is noiseless
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with chloroform-methanol-ethyl acetate-water (2.5-3-4-0.5), with this understanding, the Rf value of formononetin, hair stamen formononetin feature speckle is moderate, and it is clear to separate with other speckle, negative noiseless.
The liquid chromatograph discrimination method of formononetin, hair stamen formononetin in experimental example 11 pharmaceutical compositions
Feature for outstanding Radix Astragali flavone constituents, except the thin layer discrimination method, selected formononetin, hair stamen formononetin as its characteristic component, but because have in the pharmaceutical composition that more and formononetin, hair stamen formononetin structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and mobile phase formononetin, a hair stamen formononetin are separated:
Condition Problem
Methanol-water (70: 30) eight alkyl silane bonded silica gels Appearance time is too fast
Acetonitrile-water (50: 50) octadecylsilane chemically bonded silica Appearance time is too fast
Methanol-1% glacial acetic acid (40: 60) octadecylsilane chemically bonded silica Feminine gender has interference
Acetonitrile-0.5% formic acid (30: 70) octadecylsilane chemically bonded silica Appearance time is slow excessively
Methanol-0.1% phosphoric acid (20: 10: 70) octadecylsilane chemically bonded silica Feminine gender has interference
Acetonitrile-0.02mol/L sodium dihydrogen phosphate (30: 70) octadecylsilane chemically bonded silica Feminine gender has interference
Methanol-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water rises to 50% by 40%, from 10 minutes to 30 minutes, the ratio of water rises to 60% by 50%, from 30 minutes to 50 minutes, the ratio of water was 60% octadecylsilane keypad silica gel Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, methanol-water is a mobile phase, gradient elution, solvent ratios are that the ratio of water rose to 50% by 40% from 0 minute to 10 minutes, from 10 minutes to 30 minutes, the ratio of water rose to 60% by 50%, from 30 minutes to 50 minutes, the ratio of water is 60% for mobile phase, with this understanding, formononetin, hair stamen formononetin retention time are moderate, and the peak is capable sharp-pointed, symmetry, negative noiseless.
Scutellarin assay in experimental example 12 pharmaceutical compositions
1 instrument and reagent
1.1 key instrument:
High performance liquid chromatograph LC-2010AHT SHIMADZU
Ultraviolet/general all purpose instrument the company limited of analysing in visible spectrophotometer TU-1810SPC Beijing
Electronic analytical balance BP211D SARTORIUS
Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.
1.2 reagent:
Scutellarin Nat'l Pharmaceutical ﹠ Biological Products Control Institute
Methanol analytical pure Beijing Chemical Plant
Acetonitrile chromatographically pure Di Ma company
The pure Beijing of phosphate analysis chemical reagents corporation
2 scutellarins provide scutellarin by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and measuring purity through high performance liquid chromatography (normalization method) is 96.40%, meet assay with reference substance requirement (in mensuration, converting according to 96.40% purity).
It is an amount of that the scutellarin reference substance is got in 3 selections that detect wavelength, and accurate the title decides, and adds methanol and makes the solution that every 1ml contains 0.0463mg, in the interscan of 190～400nm wave-length coverage.The result shows that scutellarin has absorption maximum at 284nm and 335nm place, according to " the Sanitation Ministry medicine standard Chinese traditional patent formulation preparation " relevant kind and in conjunction with bibliographical information, selects the detection wavelength of 335nm as the scutellarin assay.
4 chromatographic conditions
Chromatograph: SHIMADZU LC-2010AHT;
Chromatographic column: Diamonsil ODS 250mm * 4.6mm 5 μ m;
Mobile phase: methanol-0.1% phosphate aqueous solution (50: 50);
Flow velocity: 1.0ml/min;
Column temperature: 30 ℃;
Sample size: 10 μ l;
Detect wavelength: 335nm.
It is an amount of that the preparation precision of reference substance solution takes by weighing the scutellarin reference substance, adds methanol and make the solution that every 1ml contains 0.05mg, promptly.
This product under the content uniformity item is got in the preparation of need testing solution, gets content, mixing, therefrom get about 0.25g, the accurate title, decide, and puts in the 25ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m).
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
Obtain scutellarin reference substance, test sample chromatogram according to above-mentioned chromatographic condition, its number of theoretical plate n is all greater than 2000, and the scutellarin chromatographic peak separates clear complete with close peak, and separating degree is all greater than 1.5, and solvent is noiseless.
5 linear relationships investigation precision is measured scutellarin reference substance solution (C=0.978mg/ml) 0.0,0.2,0.4,0.6,0.8,1.0ml, split in the 10ml measuring bottle, be diluted to scale with methanol, shake up, the therefrom accurate respectively 10 μ l that draw, inject chromatograph of liquid, measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).With the peak area is vertical coordinate, and the amount of scutellarin (μ g) is abscissa mapping, drawing standard curve.
Scutellarin standard curve determination result
Numbering Scutellarin amount (μ g) Conversion back (μ g) Peak area
1 2 3 4 5 0.1956 0.3912 0.5868 0.7824 0.9780 0.1886 0.3771 0.5657 0.7542 0.9428 603722 1224078 1835374 2430678 3038182
Regression equation: Y=3222232.53x+3654.50
Correlation coefficient: γ=0.9999
The result shows: scutellarin is good in 0.1886 μ g～0.9428 μ g scope internal linear.
As calculated, the standard curve of scutellarin is crossed initial point, therefore selects for use one point external standard method to measure the content of scutellarin.
Accurate scutellarin reference substance solution (0.0489mg/ml) the 10 μ l that draw of 6 precision experiment inject chromatograph of liquid, measure 5 times, investigate reference substance solution precision, and measurement result sees the following form.
The precision test
Test number (TN) 1 2 3 4 5 Meansigma methods RSD(％)
Peak area 1572015 1570202 1566206 1559384 1557017 1564965 0.42
The result shows that reference substance solution precision is good.
7 stability experiments
7.1 accurate scutellarin reference substance solution (0.0489mg/ml) the 10 μ l that draw of the stability experiment of reference substance solution inject chromatograph of liquid, inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively.
The reference substance solution stability test
Time (h) 0 2 4 8 24 Meansigma methods RSD(％)
Peak area 1572015 1570202 1566206 1559384 1557017 1564965 0.42
The result shows that 24 hours internal stabilities of reference substance solution are good.
7.2 the accurate need testing solution 10 μ l that draw of the stability experiment of need testing solution inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively.
The need testing solution stability experiment
Time (h) 0 2 4 8 24 Average RSD
Content (mg/ bottle) 2.518 2.533 2.527 2.514 2.508 2.520 0.40
The result shows that 24 hours internal stabilities of need testing solution are good.
8 repeated experiments are got this product, by operating under the preparation of text need testing solution and the algoscopy item.
Repeated experiment
Numbering 1 2 3 4 5 Meansigma methods RSD(％)
Content (mg/ bottle) 2.531 2.554 2.507 2.522 2.513 2.525 0.73
The application of sample absorption method is adopted in the experiment of 9 average recoveries, get this product under the content uniformity item, get content, mixing is therefrom got about 0.12g (totally 5 parts), the accurate title, decide, split in the 25ml measuring bottle, accurate respectively scutellarin reference substance solution (C=0.681mg/ml) 1.0ml that adds adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min, take out, put, add methanol to scale to room temperature, shake up, filter with microporous filter membrane (0.45 μ m), the accurate subsequent filtrate 10 μ l that draw inject chromatograph of liquid, measure, promptly.
The content of scutellarin is in the pharmaceutical composition: 5.755mg/g.
The average recovery experiment
Numbering Test sample sample weighting amount (g) Pure product amount (mg) in the test sample Scutellarin addition (mg) Conversion back (mg) Measured value (mg) The response rate (%)
1 2 3 4 5 0.12034 0.12207 0.12018 0.12003 0.12743 0.6926 0.7025 0.6916 0.6908 0.7334 0.681 0.681 0.681 0.681 0.681 0.6565 0.6565 0.6565 0.6565 0.6565 1.335 1.346 1.343 1.335 1.367 97.81 98.03 99.25 98.14 96.46
Average recovery rate=97.94%, RSD=1.02%
11 sample sizes are measured and are got this product three batch samples, according to the preparation of text need testing solution and the operation under the algoscopy item.
The scutellarin assay
Lot number Scutellarin content (mg/ bottle)
1 2 3 2.564 2.357 2.814
Total saponin content is measured in experimental example 13 pharmaceutical compositions
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 ginsenoside Rg1 5.14mg 2.1 detect the selection precision of wavelength, put in the 100ml measuring bottle, add an amount of methanol supersound process (power 250W, frequency 33KHz) and make dissolving, add methanol to scale, shake up, precision is measured 1.2ml, puts in the 10ml tool plug test tube water bath method solvent, take out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml shakes up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds, shake up, scan in 700～400nm wave-length coverage, the result shows that astragaloside has absorption maximum at the 547nm place, blank noiseless, therefore selecting 547nm is the detection wavelength of total saponins in the spectrophotometry 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
1 20.12 0.127
2 40.24 0.255
3 60.36 0.377
4 80.48 0.502
5 100.6 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％
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％
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％
Content (mg/ bottle) 13.26 12.89 13.34 12.76 13.18 13.09 1.90
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％
Content (mg/ bottle) 12.84 12.56 13.07 12.93 12.77 12.83 1.48
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 (0.657mg/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: 29.24mg/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 (%)
1 2 3 4 5 23.08 22.96 24.17 25.32 24.66 0.6749 0.6714 0.7067 0.7404 0.7211 0.657 0.657 0.657 0.657 0.657 1.316 1.315 1.358 1.381 1.356 97.54 98.03 99.12 97.51 96.64
Average recovery rate=97.77% RSD=0.93%
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)
1 2 3 13.51 12.42 14.37
Panax Notoginseng saponin R in experimental example 14 pharmaceutical compositions 1, the ginsenoside Rg 1, ginsenoside Rb 1Assay
1 instrument and reagent
1.1 instrument:
Alltech UVIS-201 high performance liquid chromatograph, Alltech HPLC system work station (U.S.)
KQ250B ultrasonic washing unit (Kunshan Ultrasonic Instruments Co., Ltd.)
ZK-30ABX electric vacunm drying case (in Beijing emerging great achievement instrument company)
Mettler AE240 electronic balance (Shanghai Mei Tele company)
1.2 reagent:
Panax Notoginseng saponin R 1: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
The ginsenoside Rg 1: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
Ginsenoside Rb 1: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
Used methanol, acetonitrile are chromatographically pure, and all purchasing in German Merck company water is redistilled water, and other reagent is analytical pure.
2 chromatographic conditions
Chromatographic column: Diamonsil (TM) C 18, 5 μ m; 250 * 4.6mm;
Mobile phase: acetonitrile-water is a mobile phase, gradient elution;
Gradient elution system
Time (min) Acetonitrile (%) Water (%)
0 15 21 25 20 40 20 20 80 60 80 80
Flow velocity: 1ml/min, twice sampling interval equilibration time: 3min;
Detect wavelength: 203nm;
Column temperature: 30 ℃;
Sample size: 10 μ l
Number of theoretical plate calculates by the ginsenoside Rg1 peak should be not less than 3000.
Detect the selection Panax Notoginseng saponin R of wavelength 1, the ginsenoside Rg 1, ginsenoside Rb 1, all absorption maximum is arranged at 203nm wavelength place, therefore select the detection wavelength of 203nm for use as this product.
Panax Notoginseng saponin R is got in the preparation of 3 reference substance solution 1, the ginsenoside Rg 1With ginsenoside Rb 1Reference substance is an amount of, accurate claims surely, adds methanol respectively and makes every ml and contain Panax Notoginseng saponin R 10.62mg, the ginsenoside Rg 11.42mg and ginsenoside Rb 12.14mg solution, as stock solution; Draw each 1ml of above-mentioned solution more respectively, put in same-5ml measuring bottle, add methanol, shake up, promptly get every ml and contain Panax Notoginseng saponin R to scale 10.124mg, the ginsenoside Rg 10.284mg and ginsenoside Rb 10.428mg reference substance solution.
This product under the content uniformity item is got in the preparation of 4 need testing solutions, gets content, mixing, therefrom precision takes by weighing 2.5g, puts in the 50ml measuring bottle, adds methanol 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, take out, put to room temperature, add methanol to scale, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m).
Preparation photograph this product prescription of 5 negative need testing solutions takes by weighing other composition except that Radix Notoginseng, makes negative test sample by this product preparation technology.Precision takes by weighing 2.40572g, according to the preparation below method preparation of need testing solution, as negative need testing solution.
The above-mentioned chromatographic condition of 6 system suitability experimental evidences obtains Panax Notoginseng saponin R 1, the ginsenoside Rg 1, ginsenoside Rb 1, mix reference substance, sample, negative chromatogram, number of theoretical plate n is greater than 3000, in the sample each reference substance peak separate with close peak clear fully, separating degree is greater than 1.5, and is negative noiseless.
Panax Notoginseng saponin R is got in the investigation of 7 linear relationships 1, the ginsenoside Rg 1With ginsenoside Rb 1Reference substance is an amount of, accurate claims surely, adds methanol respectively and makes every ml and contain Panax Notoginseng saponin R 10.62mg, the ginsenoside Rg 11.42mg and ginsenoside Rb 12.14mg solution, as stock solution; Precision measure aforementioned reference substance stock solution each 0.2,0.4,0.8,1.2,1.6,2.0ml, split in the 5ml measuring bottle, add methanol to scale, shake up, the therefrom accurate respectively 10 μ l that draw, inject chromatograph of liquid successively, with the peak area is vertical coordinate, and sample size (μ g) is figure for abscissa, the drawing standard curve.
Regression equation is: Panax Notoginseng saponin R 1: Y=208361.76X-1972.29 r=0.9995;
The ginsenoside Rg 1: Y=293933.01X-13982.75 r=0.9994;
Ginsenoside Rb 1: Y=192503.18X-9202.56 r=0.9993.
The result shows: Panax Notoginseng saponin R 1Good in 0.248-2.480 μ g scope internal linear relation;
The ginsenoside Rg 1Good in 0.568-5.680 μ g scope internal linear relation;
Ginsenoside Rb 1Good in 0.856-8.560 μ g scope internal linear relation.
Linear relationship test determination result
Panax Notoginseng saponin R 1X (μ g) 0.248 0.496 0.992 1.488 1.984 2.480
Peak area 50,263 100,526 200,106 305,920 413,269 511633
The ginsenoside Rg 1X (μ g) 0.568 1.136 2.272 3.408 4.544 5.680
Peak area 160,211 319,838 652,866 984,424 1,298,968 1675375
Ginsenoside Rb 1X (μ g) 0.856 1.712 3.424 5.136 6.848 8.560
Peak area 160,162 338,350 629,600 961,552 1,313,361 1650024
Reference substance mixed solution (Panax Notoginseng saponin R is got in the test of 8 precision 10.124mg/ml, the ginsenoside Rg 10.284mg/ml and ginsenoside Rb 10.428mg/ml), METHOD FOR CONTINUOUS DETERMINATION 5 times is investigated reference substance solution precision.
The precision test
Test number (TN) 1 2 3 4 5 Average RSD(％)
Panax Notoginseng saponin R 1The peak area ginsenoside Rg 1Peak area ginsenoside Rb 1Peak area 246053 742870 799153 251946 744876 790713 245960 746459 774498 249961 741460 797000 248515 737323 784495 248487 742598 789172 1.03 0.47 1.27
9 stability tests
9.1 the reference substance solution stability test is got reference substance mixed solution (Panax Notoginseng saponin R 10.124mg/ml, the ginsenoside Rg 10.284mg/ml and ginsenoside Rb 10.428mg/ml), measure at 0,2,6,10,24 hour sample introduction respectively, investigate reference substance solution stability.
The stability test of reference substance solution
Time (h) 0 2 6 10 24 Average RSD(％)
Panax Notoginseng saponin R 1The peak area ginsenoside Rg 1Peak area ginsenoside Rb 1Peak area 251645 751539 791325 250980 744648 794487 249983 752846 793693 256897 760121 787720 255921 745537 789215 253085 750938 791288 1.22 0.83 0.36
9.2 the stability test of need testing solution is got this product under the content uniformity item, gets content, mixing, and therefrom precision takes by weighing 2.30587g, by operating under the preparation of text need testing solution, measures at 0,2,6,10,24 hour sample introduction respectively.
Stability test
Time (h) 0 2 6 10 24 Average RSD(％)
Panax Notoginseng saponin R 1(mg/ bottle) ginsenoside Rg 1(mg/ bottle) ginsenoside Rb 1(mg/ bottle) 0.6713 3.521 4.526 0.6802 3.484 4.437 0.6643 3.603 4.369 0.6779 3.544 4.358 0.6812 3.507 4.406 0.6750 3.532 4.419 1.05 1.28 1.52
The result shows: need testing solution is basicly stable in 24 hours.
10 replica tests are got this product under the content uniformity item, get content, and mixing is therefrom got about 2.5g (totally 5 parts), by operating under preparation of text algoscopy need testing solution and the mensuration item.
Replica test
Numbering 1 2 3 4 5 Average RSD(％)
Panax Notoginseng saponin R 1(mg/ bottle) ginsenoside Rg 1(mg/ bottle) ginsenoside Rb 1(mg/ bottle) 0.6681 3.408 4.508 0.6712 3.513 4.603 0.6695 3.487 4.504 0.6701 3.464 4.497 0.6673 3.479 4.523 0.6692 3.470 4.527 0.23 1.13 0.96
The result shows that this law repeatability better.
11, the application of sample absorption method is adopted in average recovery test, gets this product under the content uniformity item, gets content, and mixing is therefrom got about 1.25g (totally 5 parts), puts respectively in the 50ml measuring bottle, and precision is measured mixing reference substance solution 4ml (Panax Notoginseng saponin R wherein in addition 1, the ginsenoside Rg 1And ginsenoside Rb 1Concentration be: 0.4136mg/ml, 2.3172mg/ml, 2.4408mg/ml), add an amount of supersound process of methanol (power 250W, frequency 33KHz) and make dissolving, take out, put to room temperature, add methanol to scale, shake up, filter with microporous filter membrane (0.45 μ m), the accurate 10 μ l that draw, inject chromatograph of liquid, measure, promptly.
Panax Notoginseng saponin R in the known drug compositions after measured 1, the ginsenoside Rg 1And ginsenoside Rb 1Content be 1.525,7.909 respectively, 10.318mg/g.
Panax Notoginseng saponin R 1The average recovery test
Numbering Test sample weighing (g) Pure product amount (mg) in the test sample Pure product addition (mg) Measured value (mg) The response rate (%)
1 2 3 4 5 1.21058 1.22147 1.21092 1.19983 1.20768 1.8461 1.8627 1.8467 1.8297 1.8417 1.6544 1.6544 1.6544 1.6544 1.6544 3.427 3.453 3.434 3.452 3.484 95.58 96.14 95.97 98.06 99.27
Average recovery rate=97.00%; RSD=1.64%.
Ginsenoside Rg 1The average recovery test
Numbering Test sample weighing (g) Pure product amount (mg) in the test sample Pure product addition (mg) Measured value (mg) The response rate (%)
1 2 3 4 5 1.21058 1.22147 1.21092 1.19983 1.20768 9.5745 9.6606 9.5772 9.4895 9.5515 9.2688 9.2688 9.2688 9.2688 9.2688 18.67 18.68 18.53 18.59 18.63 98.14 97.36 96.62 98.15 97.94
Average recovery rate=97.64%; RSD=0.67%.
Ginsenoside Rb 1The average recovery test
Numbering Test sample weighing (g) Pure product amount (mg) in the test sample Pure product addition (mg) Measured value (mg) The response rate (%)
1 2 3 4 5 1.21058 1.22147 1.21092 1.19983 1.20768 12.4908 12.6031 12.4943 12.3798 12.4608 9.7632 9.7632 9.7632 9.7632 9.7632 21.91 22.09 22.07 22.05 22.01 96.45 97.12 98.03 99.02 97.85
Average recovery rate=97.69%; RSD=0.99%.
12 3 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 Panax Notoginseng saponin R 1(mg/ bottle) Ginsenoside Rg 1(mg/ bottle) Ginsenoside Rb 1(mg/ bottle) Total saponins (mg/ bottle)
1 2 3 0.6484 0.6902 0.6815 3.503 3.487 3.559 4.603 4.375 4.602 8.754 8.552 8.843
Experimental example 15 Astragaloside contents are measured
Instrument and reagent
(1) key instrument:
High performance liquid chromatograph P-426 Alltech
Evaporative light scattering detector 2000ES Alltech
High performance liquid chromatograph 1100 series Agilent
Ultrasonic washing unit KQ250B Kunshan Ultrasonic Instruments Co., Ltd.
Emerging great achievement instrument company in electric vacunm drying case ZK-30ABX Beijing
Electronic analytical balance AE240 Mettler
(2) reagent: astragaloside: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
Used methanol, acetonitrile are chromatographically pure, all purchase in Tian Jinsi friend biomedical technology development company
Water is redistilled water, and other reagent is analytical pure.
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
1 chromatographic condition
Chromatographic column: Platinum C18 4.6 * 250mm 5 μ m;
Mobile phase: acetonitrile-water (32: 68);
Flow velocity: 1.0ml/min;
The evaporation photodetector detects: drift tube temperature: 110 ℃, and throughput: 2.7L/min;
Column temperature: 30 ℃.
Obtain astragaloside, test sample, negative chromatogram according to above-mentioned condition; The astragaloside peak reaches baseline separation, separates with close peak that clear fully retention time is moderate, and peak shape is sharp-pointed, symmetry, and number of theoretical plate calculates by the astragaloside peak should be not less than 2000.
The preparation of 2 reference substance solution is accurate respectively to take by weighing through 24 hours astragaloside reference substance of phosphorus pentoxide desiccator drying under reduced pressure in right amount, adds methanol and makes the mixed solution that every 1ml contains 0.2mg, promptly.
This product under the content uniformity item is got in the preparation of 3 need testing solutions, get content, porphyrize, therefrom precision takes by weighing about 5g, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml merges n-butyl alcohol liquid, uses ammonia solution thorough washing 2 times, each 40ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue add water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml successively, 40% ethanol 30ml, 70% ethanol 80ml eluting is collected 70% ethanol elution part, evaporate to dryness, residue dissolves with methanol 5ml, shake up, filter, get subsequent filtrate promptly.
4 algoscopys precision are respectively drawn reference substance solution 10 μ l, 20 μ l, and need testing solution 200 μ l inject chromatograph of liquid, measure, with the content of external standard two-point method logarithmic equation calculating astragaloside, promptly.
5 linear relationships are investigated accurate astragaloside (0.2085mg/ml) reference substance solution 2,4,8,12, the 16 μ l that draw, inject chromatograph of liquid, common logarithm value with the amount (μ g) of astragaloside is an abscissa, and the common logarithm value of peak area is that vertical coordinate is figure, the drawing standard curve.
The astragaloside linear relationship
Numbering Astragaloside (μ g) Astragaloside common logarithm value Peak area Peak area common logarithm value
1 2 3 4 5 0.417 0.834 1.668 2.502 3.336 -0.3799 -0.07883 0.2222 0.3983 0.5232 461895 971004 2022680 3111269 4312244 5.6645 5.9872 6.3059 6.4929 6.6347
Astragaloside regression equation: Y=1.0700X+6.0705;
Astragaloside correlation coefficient: γ=0.9999;
Astragaloside is good in 0.417～3.336 μ g scope internal linear.
Accurate reference substance solution (0.1668mg/ml) the 10 μ l that draw of 6 precision test inject chromatograph of liquid, continuous sample introduction 5 times.
The test of reference substance solution precision
Test number (TN) 1 2 3 4 5 Average RSD(％)
Peak area 2031539 2041338 2021467 2015966 2030855 2028233 0.48
The result shows that precision is good.
7 stability tests
7.1 accurate reference substance solution (0.1668mg/ml) the 10 μ l that draw of reference substance stability test inject chromatograph of liquid, inject chromatograph of liquid, respectively at 0,6,12,24,48 hour replication 1 time, and record peak area integrated value.
The reference substance solution stability test
Time (h) 0 6 12 24 48 Average RSD(％)
Peak area 2031539 2041338 2021467 2015966 2030855 2028233 0.48
The result shows that reference substance solution is good at 48 hours internal stabilities.
Draw same need testing solution 20 μ l 7.2 the test sample stability test is accurate, inject chromatograph of liquid, respectively 0,6,12,24,48 hour replication 1 time, record peak area integrated value.
The need testing solution stability test
Time (h) 0 6 12 24 48 Average RSD(％)
Content (mg/ bottle) 0.03188 0.03207 0.03164 0.03173 0.03205 0.03187 0.60
The result shows that need testing solution is good at 48 hours internal stabilities.
8 replica tests are got this product under the content uniformity item, get content, porphyrize, therefrom precision takes by weighing about 5g (totally 5 parts), handles by the preparation of text need testing solution and the method for measuring under the item, the therefrom accurate respectively 20 μ l that draw, inject chromatograph of liquid, measure, promptly.
Test sample replica test result
Numbering 1 2 3 4 5 Average RSD(％)
Content (mg/ bottle) 0.03321 0.03155 0.03204 0.03217 0.03249 0.03209 1.07
The result shows that repeatability is good.
The application of sample absorption method is adopted in the test of 9 average recoveries, gets this product under the content uniformity item, gets content, porphyrize, and therefrom precision takes by weighing about 2.5g (totally 5 parts), splits in the tool plug conical flask; Accurate astragaloside reference substance aqueous solution (0.1687mg/ml) 1ml (totally 6 parts) that draws, split in the above-mentioned tool plug conical flask, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml merges n-butyl alcohol liquid, uses ammonia solution thorough washing 2 times, each 40ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue add water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml successively, 40% ethanol 30ml, 70% ethanol 80ml eluting is collected 70% ethanol elution part, evaporate to dryness, residue dissolves with methanol 5ml, shake up, filter, get subsequent filtrate promptly.The accurate subsequent filtrate 20 μ l that draw inject chromatograph of liquid, measure, promptly.The content of astragaloside is respectively 0.07097mg/g in the known drug compositions after measured.
The test of astragaloside average recovery
Numbering Test sample weighing (g) Pure product amount (mg) in the test sample Pure product addition (mg) Measured value (mg) The response rate (%)
1 2 3 4 5 2.51125 2.50497 2.47906 2.48113 2.44039 0.1837 0.1832 0.1813 0.1815 0.1785 0.1687 0.1687 0.1687 0.1687 0.1687 0.3481 0.3491 0.3444 0.3456 0.3439 97.47 98.35 96.64 97.27 98.06
Astragaloside average recovery rate=97.56%; RSD=0.69%
Test agent was three batches during 10 3 batch sample Astragaloside contents were measured and got, and pressed the described method of text and handled, and the accurate 20 μ l sample introductions of drawing are measured.
Test agent Astragaloside content measurement result in three batches
Lot number Astragaloside content (mg/ bottle)
1 batch 2 batches 3 batches 0.03315 0.03271 0.03327
Formononetin, hair stamen formononetin assay in experimental example 16 pharmaceutical compositions
1 instrument and reagent
1.1 key instrument:
High performance liquid chromatograph LC-2010AHT SHIMADZU
Ultraviolet/general all purpose instrument the company limited of analysing in visible spectrophotometer TU-1810SPC Beijing
Electronic analytical balance BP211D SARTORIUS
Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.
1.2 reagent:
Methanol analytical pure Beijing Chemical Plant
Acetonitrile chromatographically pure Di Ma company
The pure Beijing of phosphate analysis chemical reagents corporation
2 formononetin, hair stamen formononetin provide formononetin, hair stamen formononetin by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, measure purity all greater than 98% through high performance liquid chromatography (normalization method), meet assay reference substance requirement.
Formononetin is got in the selections of 3 detection wavelength, hair stamen formononetin reference substance is an amount of, and accurate the title decides, and branch adds methanol and makes the solution that every 1ml contains about 0.05mg, in the interscan of 190～400nm wave-length coverage.The result shows that formononetin, hair stamen formononetin all have absorption maximum at the 250nm place, selects the detection wavelength of 250nm as formononetin, hair stamen formononetin assay.
4 chromatographic conditions
Chromatograph: SHIMADZU LC-2010AHT;
Chromatographic column: Diamonsil ODS 250mm * 4.6mm 5 μ m;
Mobile phase: methanol-water is a mobile phase, gradient elution, and solvent ratios is from 0 minute to 10 minutes, and the ratio of water is for rising to 50% from 40%, and from 10 minutes to 30 minutes, the ratio of water was for rising to 60% from 50%, and from 30 minutes to 50 minutes, the ratio of water was 60%;
Flow velocity: 1.0ml/min;
Column temperature: 30 ℃;
Sample size: 10 μ l;
Detect wavelength: 250nm.
The preparation precision of reference substance solution takes by weighing formononetin, hair stamen formononetin reference substance is an amount of, adds methanol and makes the solution that every 1ml contains 0.005mg, promptly.
This product under the content uniformity item is got in the preparation of need testing solution, gets content, mixing, therefrom get about 0.5g, the accurate title, decide, and puts in the 5ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m).
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
Obtain formononetin, hair stamen formononetin reference substance, test sample chromatogram according to above-mentioned chromatographic condition, its number of theoretical plate n is all greater than 2000, formononetin, hair stamen formononetin chromatographic peak separate clear complete with close peak, separating degree is all greater than 1.5, and solvent is noiseless.
5 linear relationships investigation precision is measured formononetin, hair stamen formononetin reference substance mixed solution (every 1ml contains formononetin 0.04808mg, hair stamen formononetin 0.04912mg respectively) 0.4,0.8,1.2,1.6,2.0ml, split in the 10ml measuring bottle, be diluted to scale with methanol, shake up, the therefrom accurate respectively 10 μ l that draw, inject chromatograph of liquid, measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).Be respectively vertical coordinate with the peak area, sample size (μ g) is abscissa mapping, drawing standard curve.
Formononetin standard curve determination result
Numbering Formononetin amount (μ g) Peak area
1 2 3 4 5 0.019232 0.038464 0.057696 0.076928 0.09616 120005 241083 360061 481146 600924
Regression equation: Y=6249485.23x+73.50
Correlation coefficient: γ=0.9999
The result shows: formononetin is good in 0.019232 μ g～0.09616 μ g scope internal linear.
Hair stamen formononetin standard curve determination result
Numbering Hair stamen formononetin amount (μ g) Peak area
1 2 3 4 5 0.019648 0.039296 0.058944 0.078592 0.09824 102151 203094 305887 407377 509362
Regression equation: Y=5184777.08x-37.30
Correlation coefficient: γ=0.9999
The result shows: hair stamen formononetin is good in 0.019648 μ g～0.09824 μ g scope internal linear.
As calculated, the standard curve of formononetin, hair stamen formononetin is all crossed initial point, therefore selects for use one point external standard method to measure the content of formononetin, hair stamen formononetin.
Accurate formononetin, hair stamen formononetin reference substance mixed solution (every 1ml contains formononetin 0.004808mg, hair stamen formononetin 0.004912mg respectively) the 10 μ l of drawing of 6 precision experiment, inject chromatograph of liquid, measure 5 times, investigate reference substance solution precision, measurement result sees the following form.
The precision test
Test number (TN) 1 2 3 4 5 Meansigma methods RSD(％)
Formononetin hair stamen formononetin 296784 255603 301152 267796 299716 255873 302058 257601 298749 259714 299692 258117 0.69 1.02
The result shows that reference substance solution precision is good.
7 stability experiments
7.1 accurate formononetin, hair stamen formononetin reference substance mixed solution (every 1ml contains formononetin 0.004808mg, hair stamen formononetin 0.004912mg respectively) the 10 μ l of drawing of the stability experiment of reference substance solution, inject chromatograph of liquid, inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively.
The test of reference substance solution stabilizing member
Time (h) 0 2 4 8 24 Meansigma methods RSD(％)
Formononetin hair stamen formononetin 296784 255603 301152 267796 299716 255873 302058 257601 298749 259714 299692 258117 0.69 1.02
The result shows that 24 hours internal stabilities of reference substance solution are good.
7.2 the accurate need testing solution 10 μ l that draw of the stability experiment of need testing solution inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively.
The need testing solution stability experiment
Time (h) 0 2 4 8 24 Average RSD％
Formononetin (mg/ bottle) hair stamen formononetin (mg/ bottle) 0.02214 0.02184 0.02137 0.02173 0.02239 0.02185 0.02185 0.02207 0.02272 0.02148 0.02209 0.02179 2.34 0.98
The result shows that 24 hours internal stabilities of need testing solution are good.
8 repeated experiments are got this product, by operating under the preparation of text need testing solution and the algoscopy item.
Repeated experiment
Numbering 1 2 3 4 5 Average RSD％
Formononetin (mg/ bottle) hair stamen formononetin (mg/ bottle) 0.02159 0.02231 0.02201 0.02209 0.02234 0.02148 0.02147 0.02227 0.02162 0.02166 0.02181 0.02196 1.65 1.70
The application of sample absorption method is adopted in the experiment of 9 average recoveries, gets this product under the content uniformity item, gets content, and mixing is therefrom got about 0.25g, and accurate the title decides, and puts in the 5ml measuring bottle; Precision is measured formononetin, hair stamen formononetin reference substance mixed solution (every 1ml contains formononetin 0.01136mg, hair stamen formononetin 0.01152mg respectively) 1ml (totally 5 parts), splits in the above-mentioned 5ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min, take out, put to room temperature, it is fixed to scale to add methanol, shake up, filter with microporous filter membrane (0.45 μ m), the accurate subsequent filtrate 10 μ l that draw inject chromatograph of liquid, measure, promptly.
The content of formononetin is in the pharmaceutical composition: 0.04971mg/g; The content of hair stamen formononetin is: 0.04937mg/g
The experiment of formononetin average recovery
Numbering Test sample sample weighting amount (g) Pure product amount (mg) in the test sample Pure product addition (mg) Measured value (mg) The response rate (%)
1 2 3 4 5 0.24478 0.25035 0.24903 0.24812 0.24754 0.01217 0.01244 0.01238 0.01233 0.01231 0.01136 0.01136 0.01136 0.01136 0.01136 0.02312 0.02359 0.02349 0.02329 0.02347 96.43 98.15 97.77 96.48 98.25
Average recovery rate=97.42%, RSD=0.92%
The experiment of hair stamen formononetin average recovery
Numbering Test sample sample weighting amount (g) Pure product amount (mg) in the test sample Pure product addition (mg) Measured value (mg) The response rate (%)
1 2 3 4 5 0.24478 0.25035 0.24903 0.24812 0.24754 0.01208 0.01236 0.01229 0.01225 0.01222 0.01152 0.01152 0.01152 0.01152 0.01152 0.02339 0.02362 0.02359 0.02321 0.02371 98.15 97.74 98.03 95.12 99.73
Average recovery rate=97.75%, RSD=1.70%
10 sample sizes are measured and are got this product three batch samples, according to the preparation of text need testing solution and the operation under the algoscopy item.
Assay
Lot number Formononetin (mg/ bottle) Hair stamen formononetin (mg/ bottle)
1 2 3 0.02205 0.02184 0.02371 0.02114 0.02036 0.02419
The specific embodiment
Embodiment 1: adopt the liquid chromatography test to be characterized as main finger printing with the Radix Astragali flavone constituents
(1) preparation of need testing solution: it is an amount of that precision takes by weighing the freeze-dried powder of this invention prescription to be measured, adds water and make the solution that every 1ml contains 1mg, shakes up, promptly;
(2) preparation of object of reference solution: get formononetin, with dissolve with methanol and be diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-water, gradient elution, solvent ratios is from 0 minute to 20 minutes, the ratio of acetonitrile is for to rise to 25% from 4%, from 20 minutes to 40 minutes, the ratio of acetonitrile is for to rise to 45% from 25%, from 40 minutes to 60 minutes, the ratio of acetonitrile is for rising to 70% from 45%, and from 60 minutes to 70 minutes, the ratio of acetonitrile was for to rise to 80% from 70%, from 70 minutes to 80 minutes, the ratio of acetonitrile is for rising to 100% from 80%, and flow velocity is 1ml/min, the detection wavelength is 254 ± 2nm, 25 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 21;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the freeze-dried powder injecta medicine compositions to be measured with the Radix Astragali flavone constituents, prepare the finger printing of freeze-dried powder to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of freeze-dried powder to be measured, meet the following requirements:
I. calculate the similarity of the finger printing and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;
II. in the freeze-dried powder finger printing to be measured, non-total peak area must not surpass 5% of total peak area;
III. the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 10%.
Embodiment 2: adopt the liquid chromatography test to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents
(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds methanol and make the solution that every 1ml contains 50mg, shakes up, promptly;
(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg 1In right amount, add methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-water, gradient elution, solvent ratios is from 0 minute to 14 minutes, the ratio of acetonitrile is 15%, and from 14 minutes to 45 minutes, the ratio of acetonitrile was for to rise to 30% from 15%, from 45 minutes to 60 minutes, the ratio of acetonitrile is for rising to 80% from 30%, and flow velocity is 1ml/min, detect wavelength 203 ± 2nm, 30 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 15;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the freeze-dried powder to be measured with Radix Astragali saponin class, arasaponin constituents, prepare the finger printing of freeze-dried powder to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of freeze-dried powder to be measured, meet the following requirements:
I. calculate the similarity of the finger printing and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;
II. in the freeze-dried powder finger printing to be measured, non-total peak area must not surpass 5% of total peak area;
III. the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 10%.
Embodiment 3: adopt the liquid chromatography test to be characterized as main finger printing with the Radix Astragali flavone constituents
(1) preparation of need testing solution: it is an amount of that precision takes by weighing prescription freeze-dried powder of the present invention, adds methanol and make the solution that every 1ml contains 1mg, shakes up, promptly;
(2) preparation of object of reference solution: get formononetin, with dissolve with methanol and be diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-1% glacial acetic acid, gradient elution, solvent ratios is from 0 minute to 20 minutes, the ratio of acetonitrile is for to rise to 22% from 3%, from 20 minutes to 45 minutes, the ratio of acetonitrile is for to rise to 46% from 22%, from 45 minutes to 65 minutes, the ratio of acetonitrile is for rising to 73% from 46%, and from 65 minutes to 75 minutes, the ratio of acetonitrile was for to rise to 81% from 73%, from 75 minutes to 80 minutes, the ratio of acetonitrile is for rising to 100% from 81%, and flow velocity is 1ml/min, the detection wavelength is 254 ± 2nm, 25 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 20;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the freeze-dried powder to be measured with the Radix Astragali flavone constituents, prepare the finger printing of freeze-dried powder to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of freeze-dried powder to be measured, meet the following requirements:
I. calculate the similarity of the finger printing and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;
II. in the freeze-dried powder finger printing to be measured, non-total peak area must not surpass 5% of total peak area;
III. the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 10%.Pharmaceutical compositions pharmaceutical compositions
Embodiment 4: adopt the liquid chromatography test to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents
(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds ethanol and make the solution that every 1ml contains 50mg, shakes up, promptly;
(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg 1In right amount, add ethanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-1% glacial acetic acid, gradient elution, solvent ratios is from 0 minute to 14 minutes, the ratio of acetonitrile is 15%, and from 14 minutes to 45 minutes, the ratio of acetonitrile was for to rise to 30% from 15%, from 45 minutes to 60 minutes, the ratio of acetonitrile is for rising to 80% from 30%, and flow velocity is 1ml/min, detect wavelength 203 ± 2nm, 30 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 13 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 13;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the freeze-dried powder to be measured with Radix Astragali saponin class, arasaponin constituents, prepare the finger printing of freeze-dried powder to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of freeze-dried powder to be measured, meet the following requirements:
I. calculate the similarity of the finger printing and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;
II. in the freeze-dried powder finger printing to be measured, non-total peak area must not surpass 5% of total peak area;
III. the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 10%.
Embodiment 5: adopt the liquid chromatography test to be characterized as main finger printing with the Radix Astragali flavone constituents
(1) preparation of need testing solution: get prescription injection 1ml of the present invention, put in the 50ml measuring bottle, add methanol, shake up, promptly to scale;
(2) preparation of object of reference solution: get formononetin, with dissolve with methanol and be diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is methanol-water, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of methanol is for to rise to 40% from 7%, from 15 minutes to 30 minutes, the ratio of methanol is for to rise to 55% from 40%, from 30 minutes to 45 minutes, the ratio of methanol is for rising to 75% from 55%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was for to rise to 85% from 75%, from 60 minutes to 40 minutes, the ratio of methanol is for rising to 100% from 85%, and flow velocity is 1ml/min, the detection wavelength is 254 ± 2nm, 25 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 22;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the injection pharmaceutical composition to be measured with the Radix Astragali flavone constituents, prepare the finger printing of injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, meet the following requirements:
I. calculate the similarity of the finger printing and the standard finger-print of injection to be measured, should be 0.90～1.00;
II. in the injection finger printing to be measured, non-total peak area must not surpass 5% of total peak area;
III. the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 10%.
Embodiment 6: adopt the liquid chromatography test to be characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents
(1) preparation of need testing solution: get injection as need testing solution;
(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg 1In right amount, add methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is methanol-0.1% phosphoric acid, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of methanol is 20%, and from 10 minutes to 40 minutes, the ratio of methanol was for to rise to 40% from 20%, from 45 minutes to 60 minutes, the ratio of methanol is for rising to 80% from 400%, and flow velocity is 1ml/min, detect wavelength 203 ± 2nm, 40 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches of collection of illustrative plates that test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 14;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the injection to be measured with Radix Astragali saponin class, arasaponin constituents, prepare the finger printing of injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, meet the following requirements:
I. calculate the similarity of the finger printing and the standard finger-print of injection to be measured, should be 0.90～1.00;
II. in the injection finger printing to be measured, non-total peak area must not surpass 5% of total peak area;
III. the relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 10%.
Embodiment 7: the thin layer chromatography discrimination method of scutellarin in the freeze-dried powder
Get freeze-dried powder 1g to be measured, add methanol 10ml and extract, centrifugal, get supernatant as need testing solution; Other gets the scutellarin reference substance, adds methanol and makes the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, put on same polyamide membrane with strip tape respectively, be developing solvent with methanol-ethyl acetate-formic acid at 7: 2: 1, launches, take out, dry, spray is with 2% ferric chloride alcoholic solution, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the streak of same color.
Embodiment 8: the liquid chromatograph discrimination method of scutellarin in the freeze-dried powder
Get freeze-dried powder 0.25g to be measured, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution is a mobile phase at 50%: 50%, and the detection wavelength is 335nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 9: Radix Notoginseng, ginsenoside Rg in the freeze-dried powder 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more thin layer chromatography discrimination method
Get freeze-dried powder 5g to be measured, extract with n-butyl alcohol 20ml, filter, filtrate volatilizes, and residue adds methanol 5ml dissolving, as need testing solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adds the chloroform reflux, extract,, discards chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances in ginsenoside Rg1, ginsenoside Rb1, the arasaponin R1, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: 10 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.
Embodiment 10: ginsenoside Rg in the freeze-dried powder 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more liquid chromatograph differentiate
Get freeze-dried powder 2.5g to be measured, put in the 50ml measuring bottle, add methanol 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, take out, put, add methanol to scale to room temperature, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); With the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 11: the thin layer chromatography discrimination method of the Radix Astragali in the freeze-dried powder
Get middle freeze-dried powder 5g to be measured, add ethanol 20ml and extract, filter the filtrate evaporate to dryness, residue adds 0.3% sodium hydroxide solution 10ml dissolving, filter, filtrate is extracted with ethyl acetate 20ml with hydrochloric acid condition pH value to 5～6, filter, filtrate evaporate to dryness, residue add ethyl acetate 1ml dissolving, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developing solvent with chloroform-methanol at 10: 1, launches, take out, dry, put in the ammonia steam and inspect under the smoked rearmounted ultra-violet lamp 365nm, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle.
Embodiment 12: the thin layer chromatography discrimination method of astragaloside in the freeze-dried powder
Get freeze-dried powder 5g to be measured, be added on the neutral alumina post after adding methanol 10ml dissolving, with 40% methanol 40ml eluting, collect eluent, evaporate to dryness, residue add water-saturated n-butanol extraction 3 times after adding water 10ml dissolving, each 10ml, merge n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue is with methanol 2ml dissolving, as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of 13: 7: 2 of chloroform-methanol-water is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts to reach under the ultra-violet lamp 365nm daylight under and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle.
Embodiment 13: the liquid chromatograph discrimination method of astragaloside in the freeze-dried powder
Get freeze-dried powder 5g to be measured, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml, merge n-butyl alcohol liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 40% ethanol 30ml, 70% ethanol 80ml eluting successively, collect 70% ethanol elution part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 32%: 68%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 14: the thin layer chromatography discrimination method of formononetin, hair stamen formononetin in the freeze-dried powder
Get freeze-dried powder 3g to be measured, add methanol 10ml and extract, extracting solution is as need testing solution; Other gets formononetin, hair stamen formononetin, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-ethyl acetate-water 2.5: 3: 4: 0.5 was developing solvent, launch, exhibition is taken out apart from 5cm, dries, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle.
Embodiment 15: the liquid chromatograph discrimination method of formononetin, hair stamen formononetin in the freeze-dried powder
Get freeze-dried powder 0.5g to be measured, the accurate title, decide, and puts in the 5ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 16: the assay of scutellarin in the freeze-dried powder
Get freeze-dried powder 0.25g to be measured, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution is a mobile phase at 50%: 50%, and the detection wavelength is 335nm; A bit calculate with external standard, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg.
Embodiment 17: ginsenoside Rg in the freeze-dried powder 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more assay
Get freeze-dried powder 2.5g to be measured, put in the 50ml measuring bottle, add methanol 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, take out, put, add methanol to scale to room temperature, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); With the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with external standard method or standard curve method, pharmaceutical composition to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount contains the ginsenoside Rg 1Limit must not be less than 5mg;
(2) the per unit amount contains ginsenoside Rb 1Limit must not be less than 7mg;
(3) the per unit amount contains Panax Notoginseng saponin R 1Limit must not be less than 0.5mg;
(4) the per unit amount contains the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The limit of summation must not be less than 12mg.
Embodiment 18: the assay of total saponins in the freeze-dried powder
Get freeze-dried powder 50mg to be measured, the accurate title, decide, and puts in the 25ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHZ) and make dissolving, take out, put to room temperature, add methanol, shake up to scale, precision is measured 1ml, puts in the 25ml measuring bottle water bath method, take out immediately, precision adds 5% vanillin-glacial acetic acid solution 0.5ml, and perchloric acid 2.0ml shakes up, heating is 15 minutes in 60 ℃ of water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, be reference substance with ginsenoside Rg1, 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 one point external standard method, pharmaceutical composition to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total saponins in the ginsenoside Rg1, must not be less than 20mg.
Embodiment 19: the assay of astragaloside in the freeze-dried powder
Get freeze-dried powder 5g to be measured, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml, merge n-butyl alcohol liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 40% ethanol 30ml, 70% ethanol 80ml eluting successively, collect 70% ethanol elution part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.04mg.
Embodiment 20: the assay of formononetin, hair stamen formononetin in the freeze-dried powder
Get freeze-dried powder 0.5g to be measured, the accurate title, decide, and puts in the 5ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add methanol, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 30 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, pharmaceutical composition to be measured is unit quantity to be equivalent to every day with output, and content limit should be following two:
(1) the per unit amount limit that contains formononetin must not be less than 0.02mg;
(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.02mg.
Embodiment 21: the thin layer chromatography discrimination method of scutellarin in the injection
Get injection 10ml to be measured, evaporate to dryness, residue add methanol 1ml dissolving, as need testing solution; Other gets the scutellarin reference substance, adds methanol and makes the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, respectively putting on same silica gel g thin-layer plate, with benzene-ethyl acetate-formic acid-water 7: 2: 1: 0.2 is developing solvent, launches, take out, dry, spray is with 1% aluminum chloride alcoholic solution, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.
Embodiment 22: the liquid chromatograph discrimination method of scutellarin in the injection
Get injection 5ml to be measured, put in the 25ml measuring bottle, add water and decide to shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m) to scale; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-1% glacial acetic acid aqueous solution is a mobile phase at 45%: 55%, and the detection wavelength is 335nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 23: Radix Notoginseng, ginsenoside Rg in the injection 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more thin layer chromatography discrimination method
Get injection 20ml to be measured, evaporate to dryness, residue methanol 5ml is as need testing solution; It is an amount of to get the Radix Notoginseng control medicinal material, and the reflux, extract, that adds methylene chloride discards dichloromethane solution, and residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1Reference substance adds methanol respectively and makes the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel H lamellae, with n-butyl alcohol-ethyl acetate-upper solution of 5: 1: 33 of water is developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.
Embodiment 24: ginsenoside Rg in the injection 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more liquid chromatograph differentiate
Get injection 25ml to be measured, put in the 50ml measuring bottle, add water to scale, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); With the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-1% glacial acetic acid aqueous solution is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 35 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 25: the thin layer chromatography discrimination method of the Radix Astragali in the injection
Get injection 20ml to be measured, evaporate to dryness, residue add 0.3% sodium hydroxide solution 10ml dissolving, filter, and filtrate is extracted with n-butyl alcohol 20ml with hydrochloric acid condition pH value to 5～6, filter, and filtrate evaporate to dryness, residue add methanol 1ml dissolving, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developing solvent with methylene chloride-methanol at 5: 0.8, launches, take out, dry, put in the ammonia steam and inspect under the smoked rearmounted ultra-violet lamp 365nm, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle.
Embodiment 26: the thin layer chromatography discrimination method of astragaloside in the injection
Get injection 20ml to be measured, evaporate to dryness, residue are added on the neutral alumina post after adding methanol 5ml dissolving, with 40% methanol 40ml eluting, collect eluent, evaporate to dryness, residue adds water-saturated n-butanol extraction 3 times after adding water 10ml dissolving, each 10ml merges n-butyl alcohol liquid, washes with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue is with methanol 2ml dissolving, as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of 13: 7: 2 of dichloromethane-ethanol-water is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts to reach under the ultra-violet lamp 365nm daylight under and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle.
Embodiment 27: the liquid chromatograph discrimination method of astragaloside in the injection
Get injection 20ml to be measured, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml, merge n-butyl alcohol liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 30% methanol 30ml, 65% methanol 80ml eluting successively, collect 65% methanol-eluted fractions part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.5% glacial acetic acid is a mobile phase at 30%: 70%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 28: the thin layer chromatography discrimination method of formononetin, hair stamen formononetin in the injection
Get injection 30ml to be measured, evaporate to dryness, residue add methanol 10ml and extract, and extracting solution is as need testing solution; Other gets formononetin, hair stamen formononetin, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with dichloromethane-ethanol-ethyl acetate-water 3: 4: 3: 0.5 was developing solvent, launches, take out, dry, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle.
Embodiment 29: the liquid chromatograph discrimination method of formononetin, hair stamen formononetin in the injection
Get injection 10ml to be measured, evaporate to dryness, residue makes dissolving in right amount with methanol and quantitatively is transferred in the 5ml measuring bottle, adds methanol and decides to shake up to scale, filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is an acetonitrile, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 55% from 45%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 65% from 55%, from 30 minutes to 50 minutes, the ratio of water is 65%, and the detection wavelength is 250nm, 40 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 30: the assay of scutellarin in the injection
Get injection 30ml to be measured, evaporate to dryness, residue add methanol makes dissolving also quantitatively be transferred in the 10ml measuring bottle in right amount, adds methanol and decides to shake up to scale, filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate as need testing solution; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and-1% glacial acetic acid aqueous solution is a mobile phase at 40%: 60%, and the detection wavelength is 335nm; A bit calculate with external standard, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg.
Embodiment 31: ginsenoside Rg in the injection 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1Assay
Get injection 25ml to be measured, put in the 50ml measuring bottle, add water to scale, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); With the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-1% glacial acetic acid aqueous solution is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is that 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 following several:
(1) the per unit amount contains the ginsenoside Rg 1Limit must not be less than 5mg;
(2) the per unit amount contains ginsenoside Rb 1Limit must not be less than 7mg;
(3) the per unit amount contains Panax Notoginseng saponin R 1Limit must not be less than 0.5mg;
(4) the per unit amount contains the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The limit of summation must not be less than 12mg.
Embodiment 32: the assay of total saponins in the injection
Precision is measured injection 0.8ml to be measured, puts in the 25ml measuring bottle water bath method, take out immediately, precision adds 5% vanillin-glacial acetic acid solution 1ml, perchloric acid 4ml, shake up, heating is 7 minutes in 60 ℃ of water-baths, takes out, immediately with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale, shake up, as need testing solution, with the ginsenoside Rg 1Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, 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 20mg.
Embodiment 33: the assay of astragaloside in the injection
Get injection 20ml to be measured, add water 20ml dissolving back water saturation n-butanol extraction, each 40ml, merge n-butyl alcohol liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 30% methanol 30ml, 65% methanol 80ml eluting successively, collect 65% methanol-eluted fractions part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.5% glacial acetic acid is a mobile phase at 30%: 70%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.04mg.
Embodiment 34: the assay of formononetin, hair stamen formononetin in the injection
Get injection 10ml to be measured, evaporate to dryness, residue makes dissolving in right amount with methanol and quantitatively is transferred in the 5ml measuring bottle, adds methanol and decides to shake up to scale, filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is an acetonitrile, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 55% from 45%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 65% from 55%, from 30 minutes to 50 minutes, the ratio of water is 65%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, pharmaceutical composition to be measured is unit quantity to be equivalent to every day with output, and content limit should be following two:
(1) the per unit amount limit that contains formononetin must not be less than 0.02mg;
(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.02mg.
Embodiment 35: the thin layer chromatography discrimination method of scutellarin in the tablet
Get tablet to be measured, pulverize, therefrom get about 1g, add ethyl acetate 10ml and extract, centrifugal, supernatant evaporate to dryness, residue add methanol 5ml dissolving as need testing solution; Other gets the scutellarin reference substance, adds methanol and makes the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, respectively putting on same silica gel H lamellae, with toluene-ethyl acetate-formic acid-water 7: 2: 1:: 0.5 is developing solvent, launches, take out, dry, spray is with 1% aluminum chloride alcoholic solution, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.
Embodiment 36: the liquid chromatograph discrimination method of scutellarin in the tablet
Get this product 0.5g, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of mobile phase (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add mobile phase, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.5% aqueous formic acid is a mobile phase at 45%: 55%, and the detection wavelength is 335nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 37: Radix Notoginseng, ginsenoside Rg in the tablet 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The thin layer chromatography discrimination method
Get this product powder 10g, use water dissolution, filter, the saturated n-butyl alcohol 20ml of filtrate water extracts, and filters, and filtrate volatilizes, and residue adds ethanol 5ml dissolving, as need testing solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, and the reflux, extract, that adds diethyl ether discards ether solution, and residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances in ginsenoside Rg1, ginsenoside Rb1, the arasaponin R1, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-Ethyl formate-methanol-water 10: 35: 20: 8 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.
Embodiment 38: ginsenoside Rg in the tablet 1, ginsenoside Rb 1, Panax Notoginseng saponin R liquid chromatograph differentiate
Get this product powder 10g, put in the 50ml measuring bottle, add mobile phase 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, takes out, and puts to room temperature, adds mobile phase to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate as need testing solution; With the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and-0.5% formic acid is mobile phase, gradient elution, solvent ratios is from 0 minute to 13 minutes, the ratio of acetonitrile is 18%, and from 13 minutes to 20 minutes, the ratio of acetonitrile rose to 42% by 18%, from 20 minutes to 25 minutes, the ratio of acetonitrile was 42%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 40 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 39: the thin layer chromatography discrimination method of the Radix Astragali in the tablet
Get this product powder 10g, add n-butyl alcohol 20ml and extract, filter the filtrate evaporate to dryness, residue adds 0.3% sodium hydroxide solution 10ml dissolving, filters, and filtrate is extracted with ethyl acetate 20ml with hydrochloric acid condition pH value to 5～6, filter, filtrate evaporate to dryness, residue add ethyl acetate 1ml dissolving, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developing solvent with chloroform-ethanol at 9: 0.5, launches, take out, dry, put in the ammonia steam and inspect under the smoked rearmounted ultra-violet lamp 365nm, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle.
Embodiment 40: the thin layer chromatography discrimination method of astragaloside in the tablet
Get this product 10g, add ethanol 40ml supersound process and make dissolving, take out, filter, filtrate evaporate to dryness, residue are added on the neutral alumina post after adding methanol 5ml dissolving, with 50% ethanol 50ml eluting, collect eluent, evaporate to dryness, residue adds water-saturated n-butanol extraction 3 times after adding water 10ml dissolving, and each 10ml merges n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue is with methanol 2ml dissolving, as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of 15: 5: 5 of methylene chloride-methanol-water is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts to reach under the ultra-violet lamp 365nm daylight under and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle.
Embodiment 41: the liquid chromatograph discrimination method of astragaloside in the tablet
Get this product powder pin 10g, add water 20ml dissolving back ethyl acetate extraction, each 40ml, merge ethyl acetate liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, ethyl acetate liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 35% ethanol 50ml, 65% ethanol 50ml eluting successively, collect 65% ethanol elution part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.5% formic acid was mobile phase in 45%: 55%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 42: the thin layer chromatography discrimination method of formononetin, hair stamen formononetin in the tablet
Get this product powder 6g, add methanol 10ml and extract, extracting solution is as need testing solution; Other gets formononetin, hair stamen formononetin, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with methylene chloride-methanol-ethyl acetate-water 3: 2: 5: 1 was developing solvent, launches, take out, dry, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle.
Embodiment 43: the liquid chromatograph discrimination method of formononetin, hair stamen formononetin in the tablet
Get this product 1g, the accurate title, decide, and puts in the 5ml measuring bottle, adds an amount of supersound process of mobile phase (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add mobile phase, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is an acetonitrile, Mobile phase B is 0.05% phosphate aqueous solution, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of Mobile phase B is for rising to 55% from 45%, and from 10 minutes to 20 minutes, the ratio of Mobile phase B was for to rise to 65% from 55%, from 30 minutes to 50 minutes, the ratio of Mobile phase B is 65%, and the detection wavelength is 250nm, 35 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
Embodiment 44: the assay of scutellarin in the tablet
Get this product 0.5g, the accurate title, decide, and puts in the 25ml measuring bottle, adds an amount of supersound process of mobile phase (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add mobile phase, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.5% aqueous formic acid is a mobile phase at 45%: 55%, and the detection wavelength is 335nm; A bit calculate with external standard, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg.
Embodiment 45: ginsenoside Rg in the tablet 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1Assay
Get this product powder 10g, put in the 50ml measuring bottle, add mobile phase 40ml, supersound process (power 250W, frequency 33KHz) makes dissolving, takes out, and puts to room temperature, adds mobile phase to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate as need testing solution; With the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The methanol solution of reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and-0.5% formic acid is mobile phase, gradient elution, solvent ratios is from 0 minute to 13 minutes, the ratio of acetonitrile is 18%, and from 13 minutes to 20 minutes, the ratio of acetonitrile rose to 42% by 18%, from 20 minutes to 25 minutes, the ratio of acetonitrile was 42%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 40 ℃; Calculate with external standard method or standard curve method, 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 5mg;
(2) the per unit amount contains ginsenoside Rb 1Limit must not be less than 7mg;
(3) the per unit amount contains Panax Notoginseng saponin R 1Limit must not be less than 0.5mg;
(4) the per unit amount contains the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The limit of summation must not be less than 12mg.
Embodiment 46: the assay of total saponins in the tablet
Get this product powder pin 0.1g, the accurate title, decide, and puts in the 25ml measuring bottle, add an amount of supersound process of ethanol (power 250W, frequency 33KHZ) and make dissolving, take out, put to room temperature, add ethanol, shake up to scale, precision is measured 1ml, put in the 25ml measuring bottle, water bath method takes out immediately, precision adds 3% vanillin-glacial acetic acid solution 1ml, perchloric acid 2.0ml shakes up, and heating is 10 minutes in 70 ℃ of water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale, shakes up immediately, as need testing solution, with the ginsenoside Rg 1Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, 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 20mg.
Embodiment 47: the assay of astragaloside in the tablet
Get this product powder pin 10g, add water 20ml dissolving back ethyl acetate extraction, each 40ml, merge ethyl acetate liquid, with ammonia solution thorough washing 2 times, each 40ml discards ammoniacal liquor, ethyl acetate liquid evaporate to dryness, residue adds water 5ml dissolving back by D101 macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), water 50ml, 35% ethanol 50ml, 65% ethanol 50ml eluting successively, collect 65% ethanol elution part, evaporate to dryness, residue shakes up with methanol 5ml dissolving, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.5% formic acid was mobile phase in 45%: 55%, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.04mg.
Embodiment 48: the assay of formononetin, hair stamen formononetin in the freeze-dried powder
Get this product powder 1g, the accurate title, decide, and puts in the 5ml measuring bottle, adds an amount of supersound process of mobile phase (power 250W, frequency 33KHz) 5min takes out, and puts to room temperature, and it is fixed to scale to add mobile phase, shake up, filter, get subsequent filtrate as need testing solution with microporous filter membrane (0.45 μ m); Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is an acetonitrile, Mobile phase B is 0.05% phosphate aqueous solution, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of Mobile phase B is for rising to 55% from 45%, and from 10 minutes to 20 minutes, the ratio of Mobile phase B was for to rise to 65% from 55%, from 30 minutes to 50 minutes, the ratio of Mobile phase B is 65%, and the detection wavelength is 250nm, 35 ℃ of column temperatures; Calculate with one point external standard method, pharmaceutical composition to be measured is unit quantity to be equivalent to every day with output, and content limit should be following two:
(1) the per unit amount limit that contains formononetin must not be less than 0.02mg;
(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.02mg.

Claims (8)

1, a kind of method of quality control of the pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali, it is characterized in that: this method comprises following all or part of content:
(1) finger printing test comprises with the Radix Astragali flavone constituents being characterized as main finger printing and being characterized as in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents;
(2) Radix Astragali control medicinal material, Radix Notoginseng control medicinal material, scutellarin, astragaloside, formononetin, calycosin, Panax Notoginseng saponin R 1, the ginsenoside Rg 1With ginsenoside Rb 1In the differential test method of all or part of composition;
(3) scutellarin, astragaloside, formononetin, calycosin, Panax Notoginseng saponin R 1, the ginsenoside Rg 1, ginsenoside Rb 1, the content test method of all or part of composition in the total saponins.
2, according to the method for quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali of claim 1, it is characterized in that: this method adopts liquid chromatography test to be characterized as main finger printing and to be characterized as in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents:
A, the test of employing liquid chromatography are characterized as main finger printing with the Radix Astragali flavone constituents:
(1) preparation of need testing solution: the 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 formononetin,, be settled to suitable concn, as object of reference solution with methanol or dissolve with ethanol;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol-water or 0.1%～5% glacial acetic acid or 0.1%～5% formic acid or 0.005%～5% phosphoric acid solution, gradient elution, flow velocity is that 0.5～2.0ml/min, detection wavelength are one or several in the 190-400nm scope, and column temperature is in 20～60 ℃ of scopes;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～50;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the pharmaceutical composition to be measured with the Radix Astragali flavone constituents, the preparation testing sample finger printing;
(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 relative retention time of total chromatographic peak of other except that the object of reference chromatographic peak and object of reference chromatographic peak and standard finger-print compare, and relative deviation must not surpass ± 20%;
B, the test of employing liquid chromatography are characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents:
(1) preparation of need testing solution: it is an amount of to get pharmaceutical composition to be measured, adds methanol or ethanol or water and is diluted to suitable concn, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Astragali and the pseudo-ginseng, comprise the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1, a kind of in the astragaloside, water or methanol or dissolve with ethanol are settled to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol-water or 0.1%～5% glacial acetic acid or 0.1%～5% formic acid or 0.005%～5% phosphoric acid solution, gradient elution, flow velocity is 0.5～2.0ml/min, detect wavelength is that one or several or evaporative light scattering detector in 190～400nm scope detects, and column temperature is in 20～60 ℃ of scopes;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～40;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the pharmaceutical composition to be measured with Radix Astragali saponin class, arasaponin constituents, the preparation testing sample finger printing;
(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 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%.
3, according to the method for quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali of claim 2, it is characterized in that: this method adopts liquid chromatography test to be characterized as main finger printing and to be characterized as in the main finger printing one or both with Radix Astragali saponin class, arasaponin constituents with the Radix Astragali flavone constituents:
A, the test of employing liquid chromatography are characterized as main finger printing with the Radix Astragali flavone constituents:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing pharmaceutical composition to be measured, adds water and make the solution that every 1ml contains 1mg, shakes up, promptly;
(2) preparation of object of reference solution: get formononetin, with dissolve with methanol and be diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-water, gradient elution, solvent ratios is from 0 minute to 20 minutes, the ratio of acetonitrile is for to rise to 25% from 4%, from 20 minutes to 40 minutes, the ratio of acetonitrile is for to rise to 45% from 25%, from 40 minutes to 60 minutes, the ratio of acetonitrile is for rising to 70% from 45%, and from 60 minutes to 70 minutes, the ratio of acetonitrile was for to rise to 80% from 70%, from 70 minutes to 80 minutes, the ratio of acetonitrile is for rising to 100% from 80%, and flow velocity is 1ml/min, the detection wavelength is 254 ± 2nm, 25 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with the Radix Astragali flavone constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the pharmaceutical composition to be measured with the Radix Astragali flavone constituents, the preparation testing sample finger printing;
(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 0.90～1.00;
II. in the pharmaceutical composition finger printing to be measured, non-total peak area must not surpass 5% of total peak area;
III. the 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%;
B, the test of employing liquid chromatography are characterized as main finger printing with Radix Astragali saponin class, arasaponin constituents:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing pharmaceutical composition to be measured, adds methanol and make the solution that every 1ml contains 50mg, shakes up, promptly;
(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg 1In right amount, add methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile-water, gradient elution, solvent ratios is from 0 minute to 14 minutes, the ratio of acetonitrile is 15%, and from 14 minutes to 45 minutes, the ratio of acetonitrile was for to rise to 30% from 15%, from 45 minutes to 60 minutes, the ratio of acetonitrile is for rising to 80% from 30%, and flow velocity is 1ml/min, detect wavelength 203 ± 2nm, 30 ℃ of column temperatures;
(4) formulation of standard finger-print: the means of testing that is characterized as main finger printing with said method as formulation with Radix Astragali saponin class, arasaponin constituents; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～20;
(5) with the described method in (1)～(3) as the means of testing that is characterized as main finger printing in the pharmaceutical composition to be measured with Radix Astragali saponin class, arasaponin constituents, the preparation testing sample finger printing;
(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 0.90～1.00;
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%.
4, according to the method for quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali of claim 1, it is characterized in that: the discrimination method of described pharmaceutical composition comprises one or more in following:
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 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 scutellarin reference substance, adds methanol or ethanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or polyamide film, with ethyl acetate or Ethyl formate-formic acid or acetic acid-water or methanol 0.2～60: 0.2～10: 0.3～20 or benzene or toluene-ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1～30: 0.5～15: 0.05～5: 0.01～3 is developing solvent, launch, take out, dry, spray is with 0.3%～10% aluminum chloride ethanol or 0.3～10% aluminum chloride methanol solution or 0.3～10% ferric chloride alcoholic solution, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect or 1～15 minute rearmounted daylight of 105 ℃ of bakings is inspected down or under ultra-violet lamp 365nm or the 254nm, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of scutellarin in b, 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 scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, with methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution 5%～95%: 95%～5% or methanol or acetonitrile-oxolane-0.01%～5% phosphate aqueous solution 2%～30%: 2%～30%: 96%～40% or acetonitrile-0.005moL/L～0.3moL/L sodium dihydrogen phosphate (1～99% phosphoric acid is transferred pH=2.0～5.0) gradient elution system is mobile phase, and the detection wavelength is 200～410nm; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
Radix Notoginseng, ginsenoside Rg in c, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more thin layer chromatography discrimination method:
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 Notoginseng control medicinal material, ginsenoside Rg 1Reference substance, ginsenoside Rb 1Reference substance, Panax Notoginseng saponin R 1In the reference substance one or more, the preparation contrast solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adding chloroform or dichloromethane or aether backflow extracts, discard chloroform or dichloromethane or ether solution, residue adds water-saturated n-butanol or ethyl acetate extraction, extracting solution adds ammonia solution, divides and gets upper strata, evaporate to dryness, residue is with methanol or dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more reference substances, add methanol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, lower floor's solution or n-butyl alcohol-ethyl acetate or the Ethyl formate-water 1～10: 0.2～2 placed below 5～40: 10～100: 5～50: 0.2～30 10 ℃ with chloroform-ethyl acetate or Ethyl formate-methanol-water: 1～15 upper strata is developing solvent, launch, take out, dry, spray is with 5～50% sulphuric acid ethanol reagent or 50% sulphate reagent, 80 ℃～160 ℃ dry by the fire to speckle colour developing clear, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color;
Ginsenoside Rg in d, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more liquid chromatograph differentiate:
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, Panax Notoginseng saponin R 1In the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution 5%～40%: 95%～60% are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
The thin layer chromatography discrimination method of the Radix Astragali in e, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, add ethyl acetate or ethanol or methanol or n-butanol extraction, filter the filtrate evaporate to dryness, residue adds the dissolving of 0.05%～5% sodium hydroxide solution, filter, filtrate is with hydrochloric acid condition pH value to 3～6, with ethyl acetate or n-butanol extraction, filter, filtrate evaporate to dryness, residue add ethyl acetate or methanol or dissolve with ethanol, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or polyamide film, with chloroform or methylene chloride-methanol or ethanol 1～30: 0.2～10 is developing solvent, launch, take out, dry, put to smoke under the rearmounted daylight or under ultra-violet lamp 365nm or the 254nm in the ammonia steam and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;
The thin layer chromatography discrimination method of astragaloside in f, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, be added on the neutral alumina post after adding methanol or dissolve with ethanol,, collect eluent with 10%～70% methanol-eluted fractions, evaporate to dryness, add water-saturated n-butanol after residue is dissolved in water and extract, merge n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae or polyamide film, lower floor's solution with chloroform or methylene chloride-methanol or alcohol-water 1～30: 1～20: 0.2～10 is developing solvent, launch, take out, dry, spray is with 2%～50% ethanol solution of sulfuric acid, 80～160 ℃ to be heated to speckle colour developing clear, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of astragaloside in g, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, and it is back with n-butyl alcohol or ethyl acetate extraction, merge extractive liquid, to be dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol or 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 15%～85%: 85%～15% methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, detecting wavelength is that 190～410nm or evaporative light scattering detector detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
The thin layer chromatography discrimination method of one or both of formononetin in h, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Other gets in formononetin, the hair stamen formononetin reference substance one or both, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, with chloroform or methylene chloride-methanol or ethanol-ethyl acetate or Ethyl formate-water 0.5～10: 1～15: 1～20: 0.1～3 is developing solvent, launch, take out, dry, put under the daylight or ultra-violet lamp 365nm or 254nm under inspect, in the test sample chromatograph, with reference substance chromatograph and the corresponding position of control medicinal material on, should show the same color speckle;
The liquid chromatograph discrimination method of one or both of formononetin in i, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Methanol or alcoholic solution with formononetin, hair stamen formononetin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, mobile phase A is methanol or acetonitrile, Mobile phase B is that water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, gradient elution, the detection wavelength is 190～410nm, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
5, according to the method for quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali of claim 4, it is characterized in that: the discrimination method of described pharmaceutical composition comprises one or more in following:
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 methanol extraction, centrifugal, gets supernatant as need testing solution; Other gets the scutellarin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 3 μ l of above-mentioned solution, put on same polyamide membrane with strip tape respectively, be developing solvent with methanol-ethyl acetate-formic acid at 7: 2: 1, launches, take out, dry, spray is with 2% ferric chloride alcoholic solution, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the streak of same color;
The liquid chromatograph discrimination method of scutellarin in b, 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 or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution is a mobile phase at 50%: 50%, and the detection wavelength is 335nm; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
Radix Notoginseng, ginsenoside Rg in c, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more thin layer chromatography discrimination method:
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 Notoginseng control medicinal material, ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more, the preparation contrast solution; The preparation of Radix Notoginseng control medicinal material solution: it is an amount of to get the Radix Notoginseng control medicinal material, adds the chloroform reflux, extract,, discards chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances in ginsenoside Rg1, ginsenoside Rb1, the arasaponin R1, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: 10 was developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;
Ginsenoside Rg in d, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more liquid chromatograph differentiate:
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, Panax Notoginseng saponin R 1In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
The thin layer chromatography discrimination method of the Radix Astragali in e, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, adds ethanol extraction, filters the filtrate evaporate to dryness, residue adds the dissolving of 0.3% sodium hydroxide solution, filters, and filtrate is used ethyl acetate extraction with hydrochloric acid condition pH value to 5～6, filter, filtrate evaporate to dryness, residue add the ethyl acetate dissolving, as need testing solution; Other gets Radix Astragali control medicinal material, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developing solvent with chloroform-methanol at 10: 1, launches, take out, dry, put in the ammonia steam and inspect under the smoked rearmounted ultra-violet lamp 365nm, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle;
The thin layer chromatography discrimination method of astragaloside in f, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, be added on the neutral alumina post after adding dissolve with methanol, use 40% methanol-eluted fractions, collect eluent, evaporate to dryness, add water-saturated n-butanol after residue is dissolved in water and extract, merge n-butyl alcohol liquid, wash with water, discard water liquid, n-butyl alcohol liquid evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other gets the astragaloside reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of 13: 7: 2 of chloroform-methanol-water is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ to be heated to speckle colour developing clear, puts to reach under the ultra-violet lamp 365nm daylight under and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of astragaloside in g, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, and back water saturation n-butanol extraction, merge extractive liquid, are dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol or 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;
The thin layer chromatography discrimination method of one or both of formononetin in h, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, and extracting solution is as need testing solution; Other gets in formononetin, the hair stamen formononetin one or both, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-ethyl acetate-water 2.5: 3: 4: 0.5 was developing solvent, launch, exhibition is taken out apart from 5cm, dries, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle;
The liquid chromatograph discrimination method of one or both of formononetin in i, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, and extracting solution is as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 20 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.
6, according to the method for quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali of claim 1, it is characterized in that: the method for testing of described pharmaceutical composition content should comprise one or more in following:
The assay of scutellarin in a, 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 scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, with methanol or acetonitrile-water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution 5%～95%: 95%～5% or or methanol or acetonitrile-oxolane-0.01%～5% phosphate aqueous solution 2%～30%: 2%～30%: 96%～40% or acetonitrile-0.005moL/L～0.3moL/L sodium dihydrogen phosphate (1～99% phosphoric acid is transferred pH=2.0～5.0) gradient elution system be mobile phase, the detection wavelength is 200～410nm; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 2mg;
Ginsenoside Rg in b, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more assay:
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, Panax Notoginseng saponin R 1In the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.5%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.5%～5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with external standard method or standard curve method, 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 per unit amount contains the ginsenoside Rg 1Limit must not be less than 2.5mg;
The per unit amount contains ginsenoside Rb 1Limit must not be less than 3.5mg;
The per unit amount contains Panax Notoginseng saponin R 1Limit must not be less than 0.25mg;
The per unit amount contains the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The limit of summation must not be less than 6mg;
The assay of total saponins in c, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, put in the measuring bottle, adding distil water makes dissolving in right amount and decides and shakes up to scale, and precision is measured in right amount, put in the measuring bottle, water bath method takes out immediately, and precision adds 1%～50% vanillin, one glacial acetic acid solution, 0.1～10ml, perchloric acid 0.1～15ml, shake up, heated 3～50 minutes in 30～80 ℃ of water-baths, take out, immediately with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale, shakes up, as need testing solution, with astragaloside or ginsenoside Rg1 or ginsenoside Rb1 or arasaponin R1 is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures trap, calculate with one point external standard method or standard curve method, 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 astragaloside or ginsenoside Rg 1Or ginsenoside Rb 1Or Panax Notoginseng saponin R 1Meter must not be less than 10mg;
The assay of astragaloside in d, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, and it is back with n-butyl alcohol or ethyl acetate extraction, merge extractive liquid, to be dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 15%～85%: 85%～1 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 are mobile phase, detecting wavelength is that 190～410nm or evaporative light scattering detector detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.02mg;
The assay of one or both of formononetin in e, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol or ethanol extraction, and extracting solution is as need testing solution; Methanol or alcoholic solution with formononetin, hair stamen formononetin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, mobile phase A is methanol or acetonitrile, Mobile phase B is that water or 0.05%～10% glacial acetic acid aqueous solution or 0.05%～10% aqueous formic acid or 0.01%～5% phosphate aqueous solution are mobile phase, gradient elution, the detection wavelength is 190～410nm, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, 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 two:
(1) the per unit amount limit that contains formononetin must not be less than 0.01mg;
(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.01mg.
7, according to the method for quality control of the described pharmaceutical composition made from breviscapine, Radix Notoginseng, the Radix Astragali 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 scutellarin in a, 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 or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-0.1% phosphate aqueous solution is a mobile phase at 50%: 50%, and the detection wavelength is 335nm; A bit calculate with external standard, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 4mg;
Ginsenoside Rg in b, the pharmaceutical composition 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1In one or more assay:
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, Panax Notoginseng saponin R 1In one or more reference substances methanol solution for the contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 15 minutes, the ratio of acetonitrile is 20%, and from 15 minutes to 21 minutes, the ratio of acetonitrile rose to 40% by 20%, from 21 minutes to 25 minutes, the ratio of acetonitrile was 20%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with external standard method or standard curve method, 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 5mg;
(2) the per unit amount contains ginsenoside Rb 1Limit must not be less than 7mg;
(3) the per unit amount contains Panax Notoginseng saponin R 1Limit must not be less than 0.5mg;
(4) the per unit amount contains the ginsenoside Rg 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1The limit of summation must not be less than 12mg;
The assay of total saponins in c, the pharmaceutical composition:
It is an amount of to get medicine to be measured, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision measures 0.6, puts in the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with the ginsenoside Rg 1Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, 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 20mg;
The assay of astragaloside in d, the pharmaceutical composition:
It is an amount of to get pharmaceutical composition to be measured, and back water saturation n-butanol extraction, merge extractive liquid, are dissolved in water, with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue is dissolved in water the back by the D101 macroporous adsorptive resins, water, 40% ethanol, 70% ethanol elution are collected 70% ethanol elution part, evaporate to dryness successively, residue is with dissolve with methanol or be diluted to suitable concn, shake up, filter, get subsequent filtrate as need testing solution; Methanol or alcoholic solution with the astragaloside reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and 32%: 68% acetonitrile-water is a mobile phase, and evaporative light scattering detector detects, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains astragaloside must not be less than 0.04mg;
The assay of one or both of formononetin in e, the pharmaceutical composition, hair stamen formononetin:
It is an amount of to get pharmaceutical composition to be measured, adds methanol extraction, and extracting solution is as need testing solution; Methanol solution with formononetin, hair stamen formononetin reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, mobile phase A is a methanol, and Mobile phase B is a water, gradient elution, solvent ratios is from 0 minute to 10 minutes, the ratio of water is for rising to 50% from 40%, and from 10 minutes to 30 minutes, the ratio of water was for to rise to 60% from 50%, from 30 minutes to 50 minutes, the ratio of water is 60%, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, 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 two:
(1) the per unit amount limit that contains formononetin must not be less than 0.02mg;
(2) the per unit amount limit that contains people Mao Rui formononetin must not be less than 0.02mg.
8, according to the method for quality control of claim 6 or the 7 described pharmaceutical compositions made from breviscapine, Radix Notoginseng, the Radix Astragali, it is characterized in that: the assay result of the ejection preparation that described pharmaceutical composition is made, calculate scutellarin, astragaloside, formononetin, calycosin, ginsenoside Rg according to percentage by weight 1, ginsenoside Rb 1, Panax Notoginseng saponin R 1, all or part of material in the total saponins total content account for deduction adjuvant and outer more than 25% of total solid of moisture in the preparation.
CNA2005101093964A 2005-10-19 2005-10-19 Quality control method of a pharmaceutical composition Pending CN1951417A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102012405A (en) * 2010-09-16 2011-04-13 陕西省中医药研究院汉唐制药有限公司 Detection method for flavonoids compounds in cotton rose general flavone
CN104422663A (en) * 2013-08-29 2015-03-18 洛阳惠中兽药有限公司 Quality inspection method of Wujiaqi oral liquid (Chinese patent medicine prepared from acanthopanax root and radix astragali)
CN110702822A (en) * 2019-02-25 2020-01-17 广州白云山医药集团股份有限公司白云山何济公制药厂 Method for detecting content of pseudo-ginseng in pseudo-ginseng traumatic rheumatism ointment
CN111366549A (en) * 2020-03-24 2020-07-03 广西壮族自治区人民医院 Method for measuring content of total saponins in red kiwi fruits

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102012405A (en) * 2010-09-16 2011-04-13 陕西省中医药研究院汉唐制药有限公司 Detection method for flavonoids compounds in cotton rose general flavone
CN102012405B (en) * 2010-09-16 2012-10-17 陕西省中医药研究院汉唐制药有限公司 Detection method for flavonoids compounds in cotton rose general flavone
CN104422663A (en) * 2013-08-29 2015-03-18 洛阳惠中兽药有限公司 Quality inspection method of Wujiaqi oral liquid (Chinese patent medicine prepared from acanthopanax root and radix astragali)
CN110702822A (en) * 2019-02-25 2020-01-17 广州白云山医药集团股份有限公司白云山何济公制药厂 Method for detecting content of pseudo-ginseng in pseudo-ginseng traumatic rheumatism ointment
CN110702822B (en) * 2019-02-25 2020-08-04 广州白云山医药集团股份有限公司白云山何济公制药厂 Method for detecting content of pseudo-ginseng in pseudo-ginseng traumatic rheumatism ointment
CN111366549A (en) * 2020-03-24 2020-07-03 广西壮族自治区人民医院 Method for measuring content of total saponins in red kiwi fruits

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