CN101991661A - Method for detecting Chinese patent drug containing at least two of white paeony root, ginseng, salvia miltiorrhiza, sweet wormwood, liquorice and angelica sinensis - Google Patents
Method for detecting Chinese patent drug containing at least two of white paeony root, ginseng, salvia miltiorrhiza, sweet wormwood, liquorice and angelica sinensis Download PDFInfo
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Abstract
The invention provides a method for detecting a Chinese patent drug containing at least two of white paeony root, ginseng, salvia miltiorrhiza, sweet wormwood, liquorice and angelica sinensis, comprising the steps as follows: preparing a sample solution and a comparison product drug solution, adopting acetonitrile and water as mobile phases, and performing high performance liquid chromatography (HPLC) measurement, wherein the detection wavelength is 201-290nm; and detecting whether a corresponding chromatographic peak exists at a position, which has the same reservation time with the chromatograph of a comparison drug, in the chromatography of a sample. Compared with the traditional thin-layer chromatography identification method, the detection method in the invention has the advantages that the mode of processing a sample containing various drugs in a liquid phase chromatograph is changed, the mixed peak in the liquid phase chromatography except the components to be detected in the Chinese patent drug is successfully eliminated, and results are more accurate and stable.
Description
Technical field
The invention provides the detection method of effective ingredient in a kind of medicine, it is a kind of detection method of middle pharmaceutically active ingredient, refers in particular to the detection method of effective ingredient in the Chinese medicine preparation that contains two or more medicinal ingredient in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, the Radix Angelicae Sinensis.
Background technology
Chinese medicine compound is the principal mode and the means of tcm clinical practice medication, the medicine of respectively distinguishing the flavor of in the side contains the number of chemical composition again, these inherent chemical constituents are material bases of compound recipe performance drug action, but the whole curative effect that medical drugs embodied during arbitrary active component can not reflect again comprehensively in the side, this is Chinese medicine compound and the fundamental difference of chemosynthesis medicine in formulating the quality standard process, so macroscopic analysis-by-synthesis becomes the inexorable trend of Chinese medicine compound development.Chinese medicine compound has been used more than one thousand years as the marrow of Chinese medicine, and from the angle of modern study, a Chinese medicine compound has characteristics such as multicomponent, multiaction, many target spots.Record in " element is asked " the most pure virginity will be discussed greatly: " the meaning monarch of main disease, the drug in a prescription helping monarch-drug is called miniSter answers the meaning of minister to make ".Along with the modernization of Chinese medicine, international deeply development, press for set up a kind of to Chinese medicine and Chinese medicine compound product from the former method of expecting that finished product carries out quality control and its complicated ingredient is detected, finger printing arises at the historic moment.
" fingerprint " (finger printing) identifies and derives from prudence that everyone fingerprint is had nothing in common with each other in small detail.According to these difference,, can determine to differentiate everyone feature by " comparison " mode.Dna fingerprinting has appearred in the development along with modern biotechnology.By the dna fingerprinting analysis, can differentiate evaluation to organisms such as people, animal, plants.
Plant amedica uses finger printing (especially chromatographic fingerprinting) to be set something afoot the seventies in 20th century aspect quality control.Current Chinese medicine fingerprint (fringerprinting) is used the dna fingerprinting development, is a kind of comprehensive, quantifiable chromatograph identification of means.What grow up at first is chemical composition of Chinese materia medica chromatographic fingerprinting, particularly high performance liquid chromatography (HPLC) finger printing.HPLC has very high separating degree, can separate the chemical constituent of complexity and form the different peak of height and form a chromatograph spy, on behalf of various different chemical compositions and its content and drug efficacy study result contact, the height of these chromatographic peaks and peak area will produce Chinese medicine spectrum effect respectively and has been learned.
A lot of Chinese patent medicines are made up of multiple flavour of a drug, for example colleague's WUJI BAIFENG WAN is made up of 19 flavor medical materials, medical material involves a wide range of knowledge, complex chemical composition, only depend on its tens active component as its quality control index, be difficult to control the inherent quality situation of Chinese patent medicine ball comprehensively, more can not make judgement its true and false quality.Therefore, utilize and to set up chromatographic fingerprinting to control its quality more objective more comprehensively than conventional criterias such as thin layer discriminating, assays.
Summary of the invention
At the defective of prior art in the existence of Chinese medicine ingredients context of detection.The inventor is through a large amount of experiments, studied the detection method of differentiating the multiple medical material of prescription in a kind of Chinese medicine preparation simultaneously, the result proves, compare with traditional thin layer chromatography discrimination method, method of the present invention avoids existing method need prepare need testing solution and control medicinal material solution respectively, the shortcoming that adopts different developing solvent systems to test respectively again, qualification result is also more objective and accurate.
Contribution of the present invention is: the inventor is applied to finger printing the unitary analysis and the evaluation of Chinese medicine preparation quality.With colleague's WUJI BAIFENG WAN is example, adopts the HPLC method to set up colleague's WUJI BAIFENG WAN (water-honeyed pill) preparation finger, for the Chinese medicine fingerprint analysis provides new thinking.
The purpose of this invention is to provide a kind of detection method that contains the Chinese patent medicine of at least two kinds of compositions in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis, can obtain result accurately, and avoided other impurity to disturb as far as possible by this method.
The invention provides a kind of detection method that contains the Chinese patent medicine of at least two kinds of compositions in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis, this method comprises:
(1). the preparation of need testing solution, take by weighing test sample, add and be equivalent to the methanol that test sample 5-10 doubly measures, dissolving, filter, filtrate is reclaimed solvent and is concentrated into driedly, and the water that residue adds with above-mentioned methanol equivalent makes dissolving, put cold, by the macroporous resin adsorption post, the 2-10 water elution doubly with above-mentioned quantity of methyl alcohol discards water liquid, reuse 15-45%wt and ethanol elution above-mentioned methanol equivalent, discard eluent, continue, collect eluent with the 2-6 ethanol elution doubly of 75-95%wt for above-mentioned quantity of methyl alcohol, evaporate to dryness, residue is with dissolve with methanol and move to measuring bottle, adds methanol to scale, crosses microporous filter membrane, get subsequent filtrate, make need testing solution;
(2). the preparation of control medicinal material solution, get at least two kinds of each 1g of control medicinal material in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis respectively, shine medical material solution with need testing solution in pairs with legal system;
(3). chromatographic condition is a mobile phase A with the acetonitrile, is Mobile phase B with water, according to the A of different proportion: the B eluting; The detection wavelength is 201-290nm;
(4). measure, draw each 10 μ L of above-mentioned solution, inject chromatograph of liquid, measure;
(5). the result detects, in the chromatograph of test sample, with the position of the identical retention time of control medicinal material chromatograph on, inspect has corresponding chromatographic peak whether respectively.
The microporous filter membrane that filters usefulness in the above-mentioned steps (1) is preferably the microporous filter membrane of 0.2～0.75 μ m, the more preferably microporous filter membrane of 0.4～0.5 μ m.
Detection method of the present invention adopts the detection method of sample process pre-treatment earlier reuse high performance liquid chromatography, changed usually containing the sample treatment of at least two kinds of medical materials in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis in the liquid chromatograph, successfully eliminated the assorted peak in the liquid phase collection of illustrative plates beyond the composition to be measured in the Chinese patent medicine preparation, the result is more precise and stable.
Above-mentioned a kind of detection method that contains the Chinese patent medicine of at least two kinds of compositions in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis provided by the invention, concrete steps comprise:
1. the preparation of need testing solution: precision takes by weighing test sample (for example 6g), add and to be equivalent to methanol that test sample 5-10 doubly measures (preferably with respect to the 6g test sample, quantity of methyl alcohol is about 50mL), dissolving, preferred supersound process (power 300W, frequency 50kHz) 30 minute, filter, filtrate is reclaimed solvent and is concentrated into dried, the water that residue adds with above-mentioned methanol equivalent makes dissolving, put cold, by D101 type macroporous resin adsorption post (the about 60g of resin of dress post, internal diameter 2cm, long 12cm), approximately being that (preferred 5-7 is doubly to methanol for the 2-10 water doubly of above-mentioned quantity of methyl alcohol, the about 50mL of quantity of methyl alcohol for example, the about 300mL of water then) eluting, discard water liquid, reuse 15-45%wt (preferred 30%wt) and ethanol (for example 300mL) eluting above-mentioned methanol equivalent, discard eluent, continue with the 2-6 that is about above-mentioned quantity of methyl alcohol (preferred 3-5 is doubly to methanol) ethanol (for example about 50mL of quantity of methyl alcohol, the then about 200mL of this ethanol) eluting doubly of 75-95%wt (preferred 80%wt), collect eluent, evaporate to dryness, residue is with dissolve with methanol and be transferred to (for example test sample is 6g, and then preferred measuring bottle is 4-6mL, more preferably the 5mL measuring bottle) in the measuring bottle, add methanol to scale, cross microporous filter membrane (microporous filter membrane of preferred 0.45 μ m), get subsequent filtrate, make need testing solution;
2. the preparation of control medicinal material solution: get at least two kinds of each 1g of control medicinal material in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, the Radix Angelicae Sinensis respectively, shine medical material solution in pairs with legal system with need testing solution;
3. chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the amount of carrying out reducing successively according to the B by 100%wt B increases the gradient elution of the amount of A until the order of the A of 100%wt; The time of gradient elution is 100-150 minute; The detection wavelength is 201-290nm, and preferred 203-180nm for example detects wavelength and is about 203nm (when detecting the Radix Paeoniae Alba, Radix Ginseng, Radix Glycyrrhizae and/or Herba Artemisiae Annuae), detects the preferably about 280nm of wavelength (when detection Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong and/or Radix Angelicae Sinensis);
4. measure: according to high performance liquid chromatography (appendix VI D) test, accurate each the 10 μ L of above-mentioned solution that draw inject chromatograph of liquid, measure;
5. the result detects: in the chromatograph of test sample, with the position of the identical retention time of control medicinal material chromatograph on, inspect has corresponding chromatographic peak whether respectively, and ultraviolet spectra is consistent.
If in the test sample chromatograph, on the position of identical with reference substance chromatograph retention time with control medicinal material, corresponding chromatographic peak is arranged respectively, and ultraviolet spectra is consistent, then can judges the medical material or the medicinal ingredient that contain reference substance in this test sample (sample); Further can convert and contain the medical material of reference substance or the content of medicinal ingredient in this test sample (sample) according to the area at peak.
Test sample involved in the present invention is meant the Chinese patent medicine (preparation) that contains at least two kinds of compositions in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis.
In the above-mentioned detection method of the present invention, the step that also preferably comprises the preparation of another contrast solution, it is the step of the preparation of reference substance solution, the preparation of this reference substance solution specifically comprises: take by weighing and the corresponding reference substance of control medicinal material, add methanol and make reference substance solution, cross 0.45 μ m microporous filter membrane, get subsequent filtrate and make reference substance solution again; This reference substance is selected from peoniflorin, liquirtin, ginsenoside Rb
1, at least a among the ginsenoside Rf, tanshinone, astragaloside, formononetin.
When above-mentioned detection method when detecting the method for the Chinese patent medicine contain the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and Radix Angelicae Sinensis, the preparation of described reference substance solution comprises that adding methanol makes every 1mL and contain peoniflorin 0.06mg/mL, liquirtin 0.02mg/mL, ginsenoside Rb respectively
10.2mg/mL, the solution of Tanshinone I I A0.016mg/mL product solution in contrast; Reference substance solution is carried out the detection of described step (3) and (4), obtain the finger printing of reference substance, then in the result of described step (5) detects, in the chromatograph of test sample, with the position of control medicinal material and the identical retention time of reference substance chromatograph on, inspect has corresponding chromatographic peak whether respectively.
With the WUJI BAIFENG WAN is example, the WUJI BAIFENG WAN (water-honeyed pill) that commodity are produced available from company of Tongrentang; The detection method of effective ingredient comprises in this WUJI BAIFENG WAN (water-honeyed pill) Chinese patent medicine:
1. the preparation of need testing solution: precision takes by weighing WUJI BAIFENG WAN (for example 6g), add the methanol (preferred 50mL) that is equivalent to test sample 5-10 and doubly measures, dissolving, preferred supersound process, filter, filtrate is reclaimed solvent and is concentrated into driedly, and the water that residue adds with above-mentioned methanol equivalent makes dissolving, put cold, by D101 type macroporous resin adsorption post, the 2-10 water elution doubly to be about above-mentioned quantity of methyl alcohol discards water liquid, the ethanol elution of the about and above-mentioned methanol equivalent of reuse 15-45%wt, discard eluent, continue, collect eluent with the 2-6 that the is about above-mentioned quantity of methyl alcohol ethanol elution doubly of 75-95%wt, evaporate to dryness, residue is with dissolve with methanol and be transferred to (for example preferred measuring bottle is 4-6mL) in the measuring bottle, adds methanol to scale, crosses microporous filter membrane (microporous filter membrane of 0.25-0.5 μ m), get subsequent filtrate, make need testing solution;
2. the preparation of control medicinal material solution: get the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, each 1g of Radix Angelicae Sinensis control medicinal material respectively, shine medical material solution in pairs with legal system with need testing solution;
3. chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the amount of carrying out reducing successively according to the B by 100%wt B increases the gradient elution of the amount of A until the order of the A of 100%wt; The time of gradient elution is 100-150 minute; The detection wavelength is 201-290nm, and preferred 203-180nm for example detects wavelength and is about 203nm (when detecting the Radix Paeoniae Alba, Radix Ginseng, Radix Glycyrrhizae and Herba Artemisiae Annuae), detects the preferably about 280nm of wavelength (when detection Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong and Radix Angelicae Sinensis);
4. measure: according to high performance liquid chromatography (appendix VI D) test, accurate each the 10 μ L of above-mentioned solution that draw inject chromatograph of liquid, measure;
5. the result detects: in the chromatograph of test sample, with the position of control medicinal material and the identical retention time of reference substance chromatograph on, inspect has corresponding chromatographic peak whether respectively, and ultraviolet spectra is consistent.
If in the test sample chromatograph, on the position of identical retention time with the control medicinal material chromatograph, corresponding chromatographic peak is arranged respectively, and ultraviolet spectra is consistent, then can judges the medical material or the medicinal ingredient that contain reference substance in this test sample (sample); Further can convert and contain the medical material of reference substance or the content of medicinal ingredient in this test sample (sample) according to the area at peak.
In the step 1 of above-mentioned detection method, it is the preparation of need testing solution, passing through D101 type macroporous resin adsorption post, with water elution, contain the carbohydrate content that contains in the test sample in the water liquid, this carbohydrate content can cause interference to the collection of illustrative plates peak of HPLC, so the present invention selects to discard water liquid, the ethanol elution of reuse 15-45%wt, trial test of the present invention shows, this part composition that elutes overlaps with the finger printing of the flow point of 80%wt ethanol elution, therefore unavoidably also will cause interference to testing result, so the present invention's selection discards the eluent of the ethanol elution of 15-45%wt, continue with the ethanol elution of 75-95%wt, repeatedly the result of trial test shows, the chromatographic peak of this part eluent is maximum, so the present invention collects the eluent of the ethanol elution of 75-95%wt.
With the WUJI BAIFENG WAN is example, the detection method of effective ingredient is the same substantially in this WUJI BAIFENG WAN (water-honeyed pill) Chinese patent medicine, the Chinese patent medicine that can detect test sample equally be the medical material that whether contains reference substance in the WUJI BAIFENG WAN (watered pill) with and component content in Chinese patent medicine.
The present invention has mainly adopted the reference substance medical material of effective ingredient in the Chinese patent medicine and reference substance (being equivalent to pure product) to make finger printing with the method for high performance liquid chromatogram, compare with the high performance liquid chromatogram collection of illustrative plates of the Chinese patent medicine preparation that contains effective ingredient again, can contrast whether contain above-mentioned effective ingredient in this Chinese patent medicine preparation.
In a preferred embodiment of the invention, be example with WUJI BAIFENG WAN (water-honeyed pill), the detection method of effective ingredient comprises in this WUJI BAIFENG WAN (water-honeyed pill) Chinese patent medicine:
1. the preparation of need testing solution: get WUJI BAIFENG WAN, porphyrize, precision takes by weighing 6g, add methanol 50mL, supersound process (power 300W, frequency 50kHz) 30 minutes, put coldly, filter, filtrate is reclaimed solvent and also is concentrated into dried, residue adds water 50mL makes dissolving, put cold, by the about 60g of D101 type macroporous resin adsorption post (internal diameter 2cm, long 12cm), with water 300mL eluting, discard water liquid, reuse 30% ethanol 300mL eluting discards eluent, continues with 80% ethanol 200mL eluting, collect eluent, evaporate to dryness, residue is with dissolve with methanol and be transferred to the 5mL measuring bottle, adds methanol to scale, shake up, cross 0.45 μ m microporous filter membrane, get subsequent filtrate, make need testing solution;
2. the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, each 1g of Radix Angelicae Sinensis control medicinal material are got in the preparation of control medicinal material solution respectively, shine medical material solution in pairs with legal system;
The preparation that also can prepare reference substance solution simultaneously: it is an amount of to get reference substance respectively, adds methanol and makes every 1mL and contain peoniflorin 0.06mg/mL, liquirtin 0.02mg/mL, ginsenoside Rb respectively
10.2mg/mL, the solution of Tanshinone I I A 0.016mg/mL, product solution in contrast;
For example get reference substance peoniflorin, liquirtin, ginsenoside Rb respectively
1, tanshinone, add methanol and make every 1mL and contain peoniflorin 0.06mg/mL, liquirtin 0.02mg/mL, ginsenoside Rb respectively
10.2mg/mL, the solution of Tanshinone I I A 0.016mg/mL, product solution in contrast;
3. chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the time of gradient elution is 100-150 minute; According to the form below carries out gradient elution; The detection wavelength is 201-205nm (preferred 203nm), main down medical material or the medicinal ingredients such as the Radix Paeoniae Alba, Radix Ginseng, Radix Glycyrrhizae, Herba Artemisiae Annuae of detecting of this wavelength, and wavelength 255-290nm, main down medical material or the medicinal ingredients such as Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, Radix Angelicae Sinensis of detecting of preferred 270-285nm, this wavelength;
Table HPLC eluent gradient
4. algoscopy: according to high performance liquid chromatography (appendix VI D) test, accurate each the 10 μ L of above-mentioned solution that draw inject chromatograph of liquid, measure.
5. the result detects: in the test sample chromatograph, with control medicinal material (or simultaneously and reference substance) the identical retention time of chromatograph position on, corresponding chromatographic peak is arranged respectively, and ultraviolet spectra is consistent.
To be the inventor set up on basis with garbled data is groped in a large amount of experiments the detection method of effective ingredient in the Chinese medicine preparation that contains two or more medicinal ingredient in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, the Radix Angelicae Sinensis simultaneously of the present invention, and described experiment is groped and the work of garbled data roughly following (is example with WUJI BAIFENG WAN (water-honeyed pill that Tongrentang produces)):
1, the foundation of analytical method and influence factor's investigation.
1.1 the selection of sample extraction condition
Different types of extraction solvent such as methanol, ethanol, water have been investigated in experiment respectively; The extraction solvent of different proportion such as 30%wt methanol, 50%wt methanol, 70%wt methanol; Different extracting method such as ultrasonic, hot reflux, Soxhlet is extracted.The result shows that methanol extraction method gained chromatographic peak is more in the chromatogram that the different proportion methanol solvate extracts.Investigated the need testing solution of n-butanol extraction and methanol extraction simultaneously, measure finger printing under above-mentioned chromatographic condition, the chromatogram of two kinds of methods does not have the difference of essence.So the methanol extraction method is adopted in this experiment, guarantees that the multiple composition in colleague's WUJI BAIFENG WAN can be embodied in the finger printing.Colleague's WUJI BAIFENG WAN is made up of 19 flavor medical materials, it is the compound preparation of a complexity, methanolic extract chromatogram peak is many but separation is not good, have nothing in common with each other according to each main effectively compositional polarity in colleague's WUJI BAIFENG WAN finished product, so investigated by high polarity to low polarity and crossed macroporous resin column, collect different proportion ethanol elution thing and separate each polar fraction and adopt different solvents (chloroform, ethyl acetate, n-butyl alcohol, methanol) Soxhlet to extract, collect two kinds of separation methods of different parts extract by the paramount polarity of low polarity.Chromatographic peak is overlapping serious in the different parts Soxhlet extract gained chromatogram, and the baseline injustice, and chromatographic peak can not obtain fine separation.Adopted macroporous resin column method gained chromatographic peak many and separate good.After the chromatographic peak feature of having investigated different proportion ethanol elution thing, finally determined the ethanol elution liquid proportional.
1.2 the selection of macroporous resin type
Adopt D-101 type macroporous resin and AB-8 type macroporous resin sample separation.The latter separates saponins chemical constituent poor effect, and the test sample saponin active ingredient is more, so select D-101 type macroporous resin.
1.3 the optimization of mobile phase and type of elution
Colleague's WUJI BAIFENG WAN ingredient is more, and nature difference is bigger, adopt the mode of isocratic elution be difficult at short notice with its effective ingredient eluting with separate, thereby adopt the gradient elution program to separate composition in colleague's WUJI BAIFENG WAN.In the selection of flow phase system,, mostly be acetonitrile-water, acetonitrile-acid system according to raw medicinal material effective ingredient classification and proportion.So investigated acetonitrile-water, acetonitrile-0.5% glacial acetic acid, acetonitrile-0.1% glacial acetic acid, acetonitrile-0.1% formic acid gradient elution chromatography system respectively.The result shows that in acetonitrile-acid system, the noise jamming of chromatographic peak baseline is serious; In the acetonitrile-water system, chromatographic peak is separated preferably, and baseline is steady.So adopt the acetonitrile-water system as mobile phase.
1.4 detect the selection of wavelength
Under the above-mentioned chromatographic condition, in 200～400nm scope need testing solution is carried out spectral scan, 3D chromatogram per sample is to choose the detection wavelength that can provide maximum chromatographic peak information.And through testing all the main fingerprint peakses of chromatographic fingerprinting that are difficult to keep this compound recipe under the discovery single wavelength.Many, comparatively concentrated at 203nm and 280nm place chromatographic peak information, baseline is more steady, and collection of illustrative plates has kept the main fingerprint peaks of other wavelength under these two wavelength.So optimum is chosen 203nm and 280nm wavelength as detecting wavelength, also according to kind, batch difference of Chinese patent medicine, and difference in experiment conditions, the wavelength that also can select 201-290nm is as detecting wavelength.
1.5 the selection of object of reference
The chromatographic peak of a composition under 280nm in the Radix Salviae Miltiorrhizae separates well with other chromatographic peaks, and retention time is suitable, and the peak type is stable; The peak purity inspection shows that this chromatographic peak is an one-component, so 280nm selects this peak as the reference peak down, all the other each chromatographic peaks all relatively calculate relative retention time and relative peak area ratio with it.The chromatographic peak of a composition under 203nm in the Radix Glycyrrhizae separates well with other chromatographic peaks, and the peak type is stable; The peak purity inspection shows that this chromatographic peak is an one-component, so 203nm selects this peak as the reference peak down, all the other each chromatographic peaks all relatively calculate relative retention time and relative peak area ratio with it.
When not containing Radix Salviae Miltiorrhizae and/or Radix Glycyrrhizae in the test sample, also can be single, separate well according to component, the peak type is stable etc., and standard is carried out examination, picks out suitable chromatographic peak as the reference peak.
1.6 assay method
Draw need testing solution 10 μ L, inject the HPLC chromatograph, other draws methanol solvate 10 μ L and injects HPLC as blank solution, and the result shows under 203nm solvent peak is arranged but do not disturb the chromatographic peak ownership, and solvent-free peak disturbs under the 280nm.
2, methodological study
2.1 precision experiment:
Get colleague's WUJI BAIFENG WAN (water-honeyed pill, lot number 4030220), the preparation need testing solution, continuous sample introduction 6 times records each total peak relative retention time and relative peak area.
Each total peak relative retention time is stable, and the RSD of each total peak relative peak area is less than 3%.The pertinent regulations that meet " specification requirement (provisional) of Chinese medicine finger printing research " precision test.
2.2 replica test:
Get 6 parts of same lot number (4030220) colleague's WUJI BAIFENG WAN (water-honeyed pill), the accurate title, decide, and is equipped with need testing solution by " 2 " below legal system, and sample introduction records each total peak relative retention time and relative peak area respectively.
Each total peak relative retention time is stable, and the RSD of each total peak relative peak area meets the pertinent regulations of " specification requirement (provisional) of Chinese medicine finger printing research " replica test less than 3%.
2.3 stability test is got same need testing solution (the same), respectively 0,4,8,12,16,20, the 24h sample introduction records each total peak relative retention time and relative peak area.
By colleague's WUJI BAIFENG WAN (water-honeyed pill) sample HPLC finger printing be studies show that it is feasible setting up colleague's WUJI BAIFENG WAN (water-honeyed pill) HPLC fingerprint spectrum method.This method is stable, favorable reproducibility, can be used as one of standard of colleague's WUJI BAIFENG WAN quality control.
3, the drafting of colleague's WUJI BAIFENG WAN HPLC finger printing and feature chromatographic peak ultraviolet spectra are pointed out
3.1 the drafting of finger printing.
Accurate colleague's WUJI BAIFENG WAN need testing solution 10 μ L that draw inject the HPLC instrument, measure the record chromatogram by above-mentioned condition.The results are shown in Figure 1 and Fig. 2.
The finger printing of other control medicinal materials is owed to give temporarily.
Except special mark and explanation, the concentration of other solvents (comprising percent concentration %) is mass concentration (%wt) among the present invention.
The Chinese patent medicine that the present invention detected is to contain in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis at least two kinds Chinese patent medicine preparation, be preferably the Chinese patent medicine preparation that contains the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and Radix Angelicae Sinensis, comprise the Chinese medicine preparation of WUJI BAIFENG WAN and/or the dosage changing form on the WUJI BAIFENG WAN basis, the Chinese medicine preparation of this dosage changing form comprises the preparation of the Chinese prescription that contains WUJI BAIFENG WAN.The Chinese patent medicine that for example the present invention detected comprises the water-honeyed pill of WUJI BAIFENG WAN, the watered pill, WUJI BAIFENG capsule and/or the WUJI BAIFENG KOUFUYE of WUJI BAIFENG WAN.
Detection method of the present invention adopts the detection method of sample process pre-treatment earlier reuse high performance liquid chromatography, changed usually containing the sample treatment of the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and/or Radix Angelicae Sinensis medical material in the liquid chromatograph, successfully eliminated the assorted peak in the liquid phase collection of illustrative plates beyond the composition to be measured in the preparation, novelty technology of the present invention especially is successfully to have developed carries out one-time detection to the Chinese patent medicine preparation that contains multidigit Chinese medicine and can contrast the technology that obtains testing result, and makes testing result more precise and stable.
Description of drawings
Fig. 1: the HPLC finger printing of colleague's WUJI BAIFENG WAN (water-honeyed pill) (detecting wavelength 203nm).
Fig. 2: the HPLC finger printing of colleague's WUJI BAIFENG WAN (water-honeyed pill) (detecting wavelength 280nm).
Wherein, marked label on Fig. 1 and Fig. 2 and be totally 19 peaks of 1～19, this 19 peaks and relatively and the relative retention time at No. 17 peaks formed the HPLC finger printing of colleague's WUJI BAIFENG WAN (water-honeyed pill) jointly.
The specific embodiment
Describe the present invention in detail below in conjunction with drawings and Examples, but do not limit practical range of the present invention.
Embodiment one. the quality determining method of colleague's WUJI BAIFENG WAN (big honeyed pills, Tongrentang's pharmacy)
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the acetonitrile is mobile phase A, is Mobile phase B with water, and according to the form below carries out gradient elution; The detection wavelength is 203nm (Radix Paeoniae Alba, Radix Ginseng, Radix Glycyrrhizae, a Herba Artemisiae Annuae medical material), 280nm (Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, Radix Angelicae Sinensis medical material).
Table HPLC eluent gradient
The preparation of need testing solution: it is an amount of to get this product, shreds, and adds equivalent kieselguhr and grinds well, precision takes by weighing 12g, adds methanol 50mL, supersound process (power 300W, frequency 50kHz) 30 minutes, put coldly, filter, filtrate is reclaimed solvent and is concentrated into dried, residue adds water 50mL makes dissolving, put cold, by the about 60g of D101 type macroporous resin adsorption post (internal diameter 2cm, long 12cm), with water 300mL eluting, discard water liquid, reuse 30% ethanol 300mL eluting, discard eluent, continue with 80% ethanol 200mL eluting, collect eluent, evaporate to dryness, residue is with dissolve with methanol and be transferred to the 5mL measuring bottle, add methanol to scale, shake up, cross 0.45 μ m microporous filter membrane, get subsequent filtrate, make need testing solution.
The preparation of control medicinal material solution: get the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, each 1g of Radix Angelicae Sinensis control medicinal material respectively, shine medical material solution in pairs with legal system.
Algoscopy: according to high performance liquid chromatography (appendix VI D) test, accurate each the 10 μ L of above-mentioned solution that draw inject chromatograph of liquid, measure the record chromatogram.
In the test sample chromatograph, on the position of identical retention time with the control medicinal material chromatograph, corresponding chromatographic peak is arranged respectively, and ultraviolet spectra is consistent.
The drafting of finger printing: the HPLC finger printing of colleague's WUJI BAIFENG WAN (detecting wavelength 203nm) is seen Fig. 1; The HPLC finger printing of colleague's WUJI BAIFENG WAN (detecting wavelength 280nm) is seen Fig. 2.
Wherein, marked label on Fig. 1 and Fig. 2 and be totally 19 peaks of 1～19, this 19 peaks and relatively and the relative retention time at No. 17 peaks formed the HPLC finger printing of colleague's WUJI BAIFENG WAN (water-honeyed pill) jointly.No. 17 peaks and No. 6 peaks are with reference to the peak.
Embodiment two. the detection method of effective ingredient in the WUJI BAIFENG WAN (watered pill, the pharmacy of remittance core)
1. the preparation of need testing solution: precision takes by weighing WUJI BAIFENG WAN (watered pill 6g), add the methanol that is equivalent to the about 8 times of amounts of test sample, dissolving, preferred supersound process 20-40 minute, filter, filtrate is reclaimed solvent and is concentrated into dried, residue adds about 50mL water makes dissolving, by D101 type macroporous resin adsorption post (the about 60g of resin of dress post, internal diameter 2cm, long 12cm), water elution with about 300mL, discard water liquid, the ethanol elution of the 50mL of reuse 25-35%wt discards eluent, the ethanol elution of about 200mL of the usefulness that continues 75-85%wt (preferred 80%wt), collect eluent, evaporate to dryness, residue is with dissolve with methanol and be transferred to (preferred 5mL measuring bottle) in the measuring bottle, add methanol to scale, cross microporous filter membrane (microporous filter membrane of preferred 0.45 μ m), get subsequent filtrate, make need testing solution;
2. the preparation of control medicinal material solution: get the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, each 1g of Radix Angelicae Sinensis control medicinal material respectively, shine medical material solution in pairs with legal system with need testing solution;
3. chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the amount of carrying out reducing successively according to the B by 100%wt B increases the gradient elution of the amount of A until the order of the A of 100%wt; The time of gradient elution is 100-150 minute; The detection wavelength is 201-290nm, preferred 203-180nm, and for example detecting wavelength is 203nm (detecting the Radix Paeoniae Alba, Radix Ginseng, Radix Glycyrrhizae and/or Herba Artemisiae Annuae), detects wavelength 280nm (detecting Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong and/or Radix Angelicae Sinensis);
4. measure: according to high performance liquid chromatography (appendix VI D) test, accurate each the 10 μ L of above-mentioned solution that draw inject chromatograph of liquid, measure;
5. the result detects: in the chromatograph of test sample, with the position of the identical retention time of control medicinal material chromatograph on, inspect has corresponding chromatographic peak whether respectively, and ultraviolet spectra is consistent.
The drafting of finger printing: the HPLC finger printing of colleague's WUJI BAIFENG WAN is seen Fig. 1 and Fig. 2.Marked 19 peaks on Fig. 1 and Fig. 2, the relative retention time at these 19 peaks and relative and No. 17 peaks has been formed the HPLC finger printing of WUJI BAIFENG WAN (water-honeyed pill) jointly, can be used as the finger printing of the contrast usefulness of WUJI BAIFENG WAN (watered pill) equally.
In the test sample chromatograph, on the position of identical retention time with the control medicinal material chromatograph, corresponding chromatographic peak is arranged respectively, and ultraviolet spectra is consistent, then can judges and contain the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, Radix Angelicae Sinensis control medicinal material or medicinal ingredient in this test sample (sample).
Embodiment three. and other contain the detection method of effective ingredient in the Chinese medicine preparation of two or more medicinal ingredient in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, the Radix Angelicae Sinensis
1. the preparation of need testing solution: precision takes by weighing test sample (should contain the Radix Paeoniae Alba in this test sample, Radix Ginseng, Radix Salviae Miltiorrhizae, 6g), add the methanol that is equivalent to the about 8 times of amounts of test sample, dissolving, filter, filtrate recovery solvent is concentrated into dried, and residue adds about 50mL water makes dissolving, by D101 type macroporous resin adsorption post (the about 60g of resin of dress post), water elution with about 300mL, discard water liquid, the ethanol elution of the 50mL of reuse 25-35%wt discards eluent, the ethanol elution of about 200mL of the usefulness that continues 75%wt, collect eluent, evaporate to dryness, residue is with dissolve with methanol and be transferred to (5mL) in the measuring bottle, add methanol to scale, cross microporous filter membrane (microporous filter membrane of preferred 0.45 μ m), get subsequent filtrate, make need testing solution;
2. the preparation of control medicinal material solution: get the Radix Paeoniae Alba, Radix Ginseng, each 1g of Radix Salviae Miltiorrhizae control medicinal material respectively, shine medical material solution in pairs with legal system with need testing solution;
3. chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the amount of carrying out reducing successively according to the B by 100%wt B increases the gradient elution of the amount of A until the order of the A of 100%wt; The time of gradient elution is 100-150 minute; The detection wavelength is 201-290nm, and preferred 203-180nm for example detects wavelength and is about 203nm (when detecting the Radix Paeoniae Alba, Radix Ginseng), detects the preferably about 280nm of wavelength (when detecting Radix Salviae Miltiorrhizae);
4. measure: according to high performance liquid chromatography (appendix VI D) test, accurate each the 10 μ L of above-mentioned solution that draw inject chromatograph of liquid, measure;
5. the result detects: in the chromatograph of test sample, with the position of control medicinal material and the identical retention time of reference substance chromatograph on, inspect has corresponding chromatographic peak whether respectively, and ultraviolet spectra is consistent.
In the test sample chromatograph, on the position of identical retention time with the control medicinal material chromatograph, corresponding chromatographic peak is arranged respectively, and ultraviolet spectra is consistent, then judges in this test sample (sample) and contain the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae.
If when control medicinal material and reference substance solution increase control medicinal material such as Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, Radix Angelicae Sinensis or medicinal ingredient, can from collection of illustrative plates, find out, do not contain Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, Radix Angelicae Sinensis medical material or medicinal ingredient in this test sample.
Embodiment four. the detection method of the effective ingredient of WUJI BAIFENG KOUFUYE (river, Jiangxi is strange)
1, instrument and reagent
Waters Alliance2695 high performance liquid chromatograph; Waters 2996 diode array detector; Waters Empower 2 data processing software systems.
B ü CHI Rotary Evaporators (Rotavapor R-114; Waterbath B-480); Lion ancient cooking vessel SHB-III circulation ability of swimming is used vacuum pump (Zhengzhou Greatwall Scientific Industrial ﹠ Trading Co., Ltd.) more; The medical numerical control supersonic washer of KQ-250DE type (Kunshan Ultrasonic Instruments Co., Ltd.); FW100 type high speed Universalpulverizer (Tianjin Tai Site Instr Ltd.).D-101 type macroporous resin (Anhui Samsung resin Science and Technology Ltd.).
Methanol, ethanol are analytical reagent; HPLC is a chromatographically pure with acetonitrile (J.T. Baker company).Water is pure water, crosses Millipore 0.22 μ m filter membrane.
Peoniflorin, liquirtin, ginsenoside Rb1, ginsenoside Rf, tanshinone, astragaloside, formononetin reference substance, the Radix Paeoniae Alba, Radix Glycyrrhizae, Herba Artemisiae Annuae, Radix Salviae Miltiorrhizae, Radix Ginseng, Rhizoma Chuanxiong, Radix Angelicae Sinensis control medicinal material are provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Colleague's WUJI BAIFENG WAN (water-honeyed pill), colleague's WUJI BAIFENG WAN feminine gender (lacking flavor) are provided by Beijing TongrenTang Co., Ltd Tongrentang Pharmaceutical Factory, Beijing.Radix Salviae Miltiorrhizae, Radix Ginseng, the Radix Paeoniae Alba, Radix Glycyrrhizae, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Herba Artemisiae Annuae, Rhizoma Cyperi medical material sample are provided by Beijing TongrenTang Co., Ltd Tongrentang Pharmaceutical Factory, Beijing, open the associate professor of pharmacy that continues through Nat'l Pharmaceutical ﹠ Biological Products Control Institute and identify.
2, the preparation of test solution product: get WUJI BAIFENG KOUFUYE 20mL, by the D101 type macroporous resin adsorption post (internal diameter 2cm, long 12cm) that pretreatment is good, with water 300mL eluting, discard water liquid, reuse 30% ethanol 300mL eluting, discard eluent, continue, collect eluent with 80% ethanol 200mL eluting, evaporate to dryness with dissolve with methanol and be transferred to the 5mL measuring bottle, adds methanol to scale, shake up, cross 0.45 μ m microporous filter membrane, get subsequent filtrate, promptly.
3, the preparation of reference substance solution: get peoniflorin, liquirtin, ginsenoside Rb respectively
1, ginsenoside Rf, Tanshinone I I A, astragaloside, formononetin reference substance be an amount of, adds methanol and make reference substance solution, crosses 0.45 μ m microporous filter membrane, gets subsequent filtrate, promptly.
4, HPLC chromatographic condition:
Chromatographic column: Alltech Alltima C
18Post (4.6mm * 250mm, 5 μ m); Mobile phase: see the following form; Flow velocity: 1mLmin
-1Detect wavelength: 203nm, 280nm; Column temperature: 25 ℃; Sample size: 10 μ L.Chromatogram writing time is 126 minutes.
Table HPLC eluent gradient
Embodiment five. result's contrast of Chinese patent medicine reference examples
The Chinese patent medicine reference examples is to have deleted the finished product that one or several effective ingredient obtain on the prescription basis of former Chinese patent medicine.Present embodiment is to have deleted the Radix Paeoniae Alba and Radix Ginseng on the prescription basis of WUJI BAIFENG WAN.
1. instrument and reagent: with embodiment one.
2. chromatographiccondition: with embodiment one.
3. the preparation of test sample (reference examples) solution: with embodiment one.
4. measure: accurate respectively reference substance solution and each 10ul of test sample (reference examples) solution of drawing, inject chromatograph of liquid, measure, promptly.
5. contrast collection of illustrative plates: the result does not contain the Radix Paeoniae Alba and Radix Ginseng in the test sample (reference examples) as can be seen, and other compositions for example Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and Radix Angelicae Sinensis then are present in the test sample (reference examples).
Result's proof is consistent with the actual composition of reference examples.
Embodiment six. the detection method of effective ingredient in the WUJI BAIFENG capsule (Beijing Tongrentang)
1. the preparation of need testing solution: precision takes by weighing WUJI BAIFENG capsule 's content 6g, add the methanol that is equivalent to the about 8 times of amounts of test sample, dissolving, preferred supersound process 20-40 minute, filter, filtrate is reclaimed solvent and is concentrated into dried, residue adds about 50mL water makes dissolving, by D101 type macroporous resin adsorption post (the about 60g of resin of dress post, internal diameter 2cm, long 12cm), water elution with about 300mL, discard water liquid, the ethanol elution of the 50mL of reuse 25-35%wt discards eluent, the ethanol elution of about 200mL of the usefulness that continues 75-85%wt (preferred 80%wt), collect eluent, evaporate to dryness, residue is with dissolve with methanol and be transferred to (preferred 5mL measuring bottle) in the measuring bottle, add methanol to scale, cross microporous filter membrane (microporous filter membrane of preferred 0.45 μ m), get subsequent filtrate, make need testing solution;
2. the preparation of control medicinal material solution: get the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, each 1g of Radix Angelicae Sinensis control medicinal material respectively, shine medical material solution in pairs with legal system with need testing solution;
3. chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the amount of carrying out reducing successively according to the B by 100%wt B increases the gradient elution of the amount of A until the order of the A of 100%wt; The time of gradient elution is 100-150 minute; The detection wavelength is 201-290nm, preferred 203-180nm, and for example detecting wavelength is 203nm (detecting the Radix Paeoniae Alba, Radix Ginseng, Radix Glycyrrhizae and/or Herba Artemisiae Annuae), detects wavelength 280nm (detecting Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong and/or Radix Angelicae Sinensis);
4. measure: according to high performance liquid chromatography (appendix VI D) test, accurate each the 10 μ L of above-mentioned solution that draw inject chromatograph of liquid, measure;
5. the result detects: in the chromatograph of test sample, with the position of the identical retention time of control medicinal material chromatograph on, inspect has corresponding chromatographic peak whether respectively, and ultraviolet spectra is consistent.
In the test sample chromatograph, on the position of identical retention time with the control medicinal material chromatograph, corresponding chromatographic peak is arranged respectively, and ultraviolet spectra is consistent, then can judges and contain the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong, Radix Angelicae Sinensis control medicinal material or medicinal ingredient in this test sample (sample).
Claims (10)
1. detection method that contains in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis at least two kinds Chinese patent medicine, this method comprises:
(1). the preparation of need testing solution, precision takes by weighing test sample, adds to be equivalent to the methanol that test sample 5-10 doubly measures, dissolving, filter, filtrate is reclaimed solvent and is concentrated into driedly, and the water that residue adds with above-mentioned methanol equivalent makes dissolving, put cold, by the macroporous resin adsorption post, the 2-10 water elution doubly with above-mentioned quantity of methyl alcohol discards water liquid, reuse 15-45%wt and ethanol elution above-mentioned methanol equivalent, discard eluent, continue, collect eluent with the 2-6 ethanol elution doubly of 75-95%wt for above-mentioned quantity of methyl alcohol, evaporate to dryness, residue is with dissolve with methanol and be transferred in the measuring bottle, adds methanol to scale, crosses microporous filter membrane, get subsequent filtrate, make need testing solution;
(2). the preparation of control medicinal material solution, get at least two kinds of each 1g of control medicinal material in the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and the Radix Angelicae Sinensis respectively, shine medical material solution with need testing solution in pairs with legal system;
(3). chromatographic condition is a mobile phase A with the acetonitrile, is Mobile phase B with water, according to the A:B eluting of different proportion; The detection wavelength is 201-290nm;
(4). measure, draw each 10 μ L of above-mentioned solution, inject chromatograph of liquid, measure;
(5). the result detects, in the chromatograph of test sample, with the position of the identical retention time of control medicinal material chromatograph on, inspect has corresponding chromatographic peak whether respectively.
2. detection method as claimed in claim 1, wherein, the concentration of described need testing solution is that every ml is equivalent to test sample 0.1-10g.
3. detection method as claimed in claim 1, wherein, the described detection wavelength of step (3) is 203-280nm.
4. detection method as claimed in claim 1, wherein, the described mobile phase of step (3) is Mobile phase B with water for being mobile phase A with the acetonitrile, reduces the amount of the amount increase A of B according to the B by 100%wt successively and carries out gradient elution until the order of the A of 100%wt.
5. detection method as claimed in claim 1, it is the detection method that contains effective ingredient in the Chinese patent medicine of the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and Radix Angelicae Sinensis, wherein, step (2) is made control medicinal material solution for getting the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and each 1g of Radix Angelicae Sinensis control medicinal material respectively.
6. detection method as claimed in claim 1, wherein, this detection method also comprises the step of the preparation of reference substance solution, being prepared as of this reference substance solution takes by weighing and the corresponding reference substance of control medicinal material, add methanol and make reference substance solution, cross 0.45 μ m microporous filter membrane, get subsequent filtrate and make reference substance solution again; This reference substance is selected from peoniflorin, liquirtin, ginsenoside Rb
1, at least a among the ginsenoside Rf, tanshinone, astragaloside, formononetin.
7. detection method as claimed in claim 6, it is the detection method that contains the Chinese patent medicine of the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and Radix Angelicae Sinensis, wherein, in the preparation of described reference substance solution, comprise that adding methanol makes every 1mL and contain peoniflorin 0.06mg/mL, liquirtin 0.02mg/mL, ginsenoside Rb respectively
10.2mg/mL, the solution of Tanshinone I I A 0.016mg/mL product solution in contrast; Reference substance solution is carried out the detection of described step (3) and (4), obtain the finger printing of reference substance, then in described step (5), in the chromatograph of test sample, with the position of control medicinal material and the identical retention time of reference substance chromatograph on, inspect has corresponding chromatographic peak whether respectively.
8. as each described detection method of claim 1-7, it is the detection method that contains the Chinese patent medicine of the Radix Paeoniae Alba, Radix Ginseng, Radix Salviae Miltiorrhizae, Herba Artemisiae Annuae, Radix Glycyrrhizae, Rhizoma Chuanxiong and Radix Angelicae Sinensis, and wherein, step (4) is to be filler with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the time of gradient elution is 100-150 minute; Detect wavelength and under 201-205nm, detect the Radix Paeoniae Alba, Radix Ginseng, Radix Glycyrrhizae, Herba Artemisiae Annuae, detect wavelength and detect Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, Radix Angelicae Sinensis at 255-290nm.
9. detection method as claimed in claim 8, wherein, the Chinese patent medicine that is detected is included in WUJI BAIFENG WAN and/or contains the Chinese medicine preparation of the Chinese prescription of WUJI BAIFENG WAN.
10. detection method as claimed in claim 9, wherein, the Chinese patent medicine that is detected comprises the water-honeyed pill of WUJI BAIFENG WAN, the watered pill, WUJI BAIFENG capsule and/or the WUJI BAIFENG KOUFUYE of WUJI BAIFENG WAN.
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| CN2009101664780A CN101991661B (en) | 2009-08-19 | 2009-08-19 | Method for detecting Chinese patent drug containing at least two of white paeony root, ginseng, salvia miltiorrhiza, sweet wormwood, liquorice and angelica sinensis |
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