CN103616459A - Finger-print detection method of traditional Chinese medicine Kuhuang injection, and raw medicinal materials and semi-finished product thereof as well as application thereof - Google Patents
Finger-print detection method of traditional Chinese medicine Kuhuang injection, and raw medicinal materials and semi-finished product thereof as well as application thereof Download PDFInfo
- Publication number
- CN103616459A CN103616459A CN201310692364.6A CN201310692364A CN103616459A CN 103616459 A CN103616459 A CN 103616459A CN 201310692364 A CN201310692364 A CN 201310692364A CN 103616459 A CN103616459 A CN 103616459A
- Authority
- CN
- China
- Prior art keywords
- kuhuang
- mobile phase
- injection
- volume ratio
- finger
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Medicines Containing Plant Substances (AREA)
- Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a finger-print detection method of a traditional Chinese medicine Kuhuang injection, and raw medicinal materials and a semi-finished product thereof as well as an application thereof. The method comprises preparation of a test liquid, establishment of a contrast finger-print and test of the finger-print of the test liquid. According to the structural properties and characteristics of active components including anthraquinones, flavonoids and alkaloids of the traditional Chinese medicine Kuhuang injection provided by the invention, the optimal mobile phase constitution and the optimal analytical conditions such as a gradient elution procedure, flow rate, detection wavelengths, a chromatographic column and column temperature are screened by a lot of experiments. Multiple experiments verify that the finger-print detection method of the traditional Chinese medicine Kuhuang injection, and the raw medicinal materials and the semi-finished product thereof can comprehensively, objectively and accurately detect and evaluate the quality of the Kuhuang injection, and further can guarantee safety and effectiveness of the raw medicinal materials of the Kuhuang injection from the source, so that the method disclosed by the invention has important meaning for guaranteeing clinical effects.
Description
Technical field
The present invention relates to a kind of quality detecting method of Chinese medicine preparation, be specifically related to a kind of " kuhuang " injection and raw medicinal material thereof, half-finished fingerprint atlas detection method and application.
Background technology
Kuhuang injection is classical traditional Chinese medicine, has stable and good clinical efficacy.Prescription comes from " Yinchenhao Tang, Oriental Wormwood Decoction " (oriental wormwood, rheum officinale, the mountain mast) in < < Treatise on Fevrile Diseases > >.This side is always acknowledged as the recipe for the treatment of jaundice with damp-heat pathogen." kuhuang " injection on this side basis, plus-minus and obtain.This injection is early than approval list marketing in 96 years, operative norm is the national drug standards (new drugs become a full member standard the 30th), traditional Chinese medicine (medicine number of registration is the accurate word Z10960004 of traditional Chinese medicines) for a kind of effective treatment icteric viral hepatitis of Changshu Leiyunshang Pharmaceutical Co., Ltd's (predecessor's State-run Changshou Pharmaceutical Factory) exploitation, Kuhuang injection is sold over nearly more than 20 years, clinical effectiveness is obvious, for hepatopath has brought the chance of rehabilitation." kuhuang " injection is made by 5 taste medicinal materials such as rheum officinale, kuh-seng, folium isatidis, HERBA BUPLEURL, oriental wormwoods, and because medicinal material is more, complex chemical composition, causes its quality control difficulty larger.Kuhuang injection active component is mainly flavonoids, Anthraquinones and alkaloid compound, but the quality standard detecting method of Kuhuang injection active component can only detect separately active component one by one at present, or can only detect separately certain position, testing is loaded down with trivial details, detection efficiency is low, and the active component limited amount detecting, the well quality of response preparation.
Therefore, necessary on the basis of prior art, design research and development are a kind of can complete detection and the fingerprint atlas detection method of evaluating " kuhuang " injection quality.
Summary of the invention
Goal of the invention: the object of the invention is to solve the deficiencies in the prior art, a kind of " kuhuang " injection and raw material thereof, half-finished fingerprint atlas detection method are provided, this detection method can be evaluated the quality of Kuhuang injection objective, comprehensively and accurately, to controlling the quality of Kuhuang injection and the quality of raw medicinal material and guaranteeing that clinical efficacy is significant.
Technical scheme: in order to realize above technical purpose, the technical scheme that the present invention takes is:
The fingerprint atlas detection method of Chinese medicine " kuhuang " injection, comprises the following steps:
(1) need testing solution preparation: get " kuhuang " injection as need testing solution;
(2) reference fingerprint of high effective liquid chromatography for measuring " kuhuang " injection: get 10 batches of above " kuhuang " injection samples sample introduction under suitable chromatographic condition, record the finger-print of each batch of " kuhuang " injection, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print in contrast;
(3) high effective liquid chromatography for measuring need testing solution finger-print: " kuhuang " injection need testing solution to be measured, by the identical chromatographic condition sample introduction of step (2), is recorded to the finger-print of need testing solution;
(4) the reference fingerprint similarity software evaluation that need testing solution finger-print step (3) being obtained and step (2) obtain.
The fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection, the described chromatographic condition of step (2) and step (3) is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 35～40 ℃ of column temperatures, detection wavelength is 250～260nm.
The fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection, the described chromatographic condition of step (2) and step (3) is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30～35 ℃ of column temperatures, detection wavelength is 260～270nm.
As preferred version, the fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection, the described chromatographic condition of step (2) and step (3) is: with DianmonsilC18 (5 μ m, 4.6 * 250mm) for mensuration chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 40 ℃ of column temperatures, detection wavelength is 254nm.Linear gradient elution mode is: the ratio of mobile phase A in the time of 0 minute: B is 85:15, and the ratio of mobile phase A in the time of 25 minutes: B is 33:67, and the ratio of mobile phase A in the time of 65 minutes: B is 0:100.
As preferred version, the fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection, the described chromatographic condition of step (2) and step (3) is: with DianmonsilC18 (5 μ m, 4.6 * 250mm) for mensuration chromatographic column, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.Linear gradient elution mode is: the ratio of mobile phase A in the time of 0 minute: B is 98:2, the ratio of mobile phase A in the time of 20 minutes: B is 84:16, the ratio of mobile phase A in the time of 30 minutes: B is 80:20, and the ratio of mobile phase A in the time of 90 minutes: B is 40:60, and the ratio of mobile phase A in the time of 100 minutes: B is 0:100.
The fingerprint atlas detection method of Chinese medicine " kuhuang " injection raw medicinal material provided by the invention, comprises the following steps:
(1) need testing solution preparation: get " kuhuang " injection raw medicinal material, obtain after treatment the need testing solution of raw medicinal material;
(2) reference fingerprint of high effective liquid chromatography for measuring raw material: get respectively " kuhuang " injection raw medicinal material respectively more than 10 batches, disposal route by step (1) makes need testing solution, under suitable chromatographic condition, record the finger-print of every batch of raw medicinal material, take these finger-prints as basis, with similarity software, calculate and obtain common pattern as raw medicinal material reference fingerprint;
(3) high effective liquid chromatography for measuring need testing solution finger-print: get the need testing solution of step (1) by the identical chromatographic condition sample introduction of step (2), record the finger-print of the need testing solution of " kuhuang " injection raw medicinal material;
(4) the reference fingerprint similarity software evaluation that need testing solution finger-print step (3) being obtained and step (2) obtain.
The fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection raw material, the raw medicinal material of " kuhuang " injection is rheum officinale, kuh-seng, oriental wormwood, HERBA BUPLEURL and folium isatidis medicinal material.
The fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection raw medicinal material, the described raw medicinal material disposal route of step (1) is: by pulverizing medicinal materials, get medicinal powder 4.0g, and accurately weighed, add water 100ml, add hot reflux 1～2 hour, filter, get subsequent filtrate 50ml and be concentrated into about 10ml, adding ethanol makes to reach 85% containing alcohol amount, placement is spent the night, and filters, and the concentrated also water of filtrate is settled to 10ml.
The fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection raw medicinal material, the described chromatographic condition of step (2) and step (3) is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 35～40 ℃ of column temperatures, detection wavelength is 250～260nm.
The fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection raw medicinal material, the described chromatographic condition of step (2) and step (3) is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30～35 ℃ of column temperatures, detection wavelength is 260～270nm.
As preferred version, the fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection raw medicinal material, the described chromatographic condition of step (2) and step (3) is: with DianmonsilC18 (5 μ m, 4.6 * 250mm) for mensuration chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 40 ℃ of column temperatures, detection wavelength is 254nm.Linear gradient elution mode is: the ratio of mobile phase A in the time of 0 minute: B is 85:15, and the ratio of mobile phase A in the time of 25 minutes: B is 33:67, and the ratio of mobile phase A in the time of 65 minutes: B is 0:100.
As preferred version, the fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection raw medicinal material, the described chromatographic condition of step (2) and step (3) is: with DianmonsilC18 (5 μ m, 4.6 * 250mm) for mensuration chromatographic column, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.Linear gradient elution mode is: the ratio of mobile phase A in the time of 0 minute: B is 98:2, the ratio of mobile phase A in the time of 20 minutes: B is 84:16, the ratio of mobile phase A in the time of 30 minutes: B is 80:20, and the ratio of mobile phase A in the time of 90 minutes: B is 40:60, and the ratio of mobile phase A in the time of 100 minutes: B is 0:100.
The half-finished fingerprint atlas detection method of Chinese medicine " kuhuang " injection provided by the invention, comprises the following steps:
(1) need testing solution preparation: get " kuhuang " injection semi-manufacture as need testing solution;
(2) the half-finished reference fingerprint of high effective liquid chromatography for measuring " kuhuang " injection: get 10 batches of above " kuhuang " injection semi-manufacture sample introduction under suitable chromatographic condition, record each batch of half-finished finger-print of " kuhuang " injection, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print in contrast;
(3) high effective liquid chromatography for measuring need testing solution finger-print: " kuhuang " injection semi-manufacture need testing solution to be measured, by the identical chromatographic condition sample introduction of step (2), is recorded to the finger-print of need testing solution;
(4) the reference fingerprint similarity software evaluation that need testing solution finger-print step (3) being obtained and step (2) obtain.
The half-finished fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection, the described chromatographic condition of step (2) and step (3) is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 35～40 ℃ of column temperatures, detection wavelength is 250～260nm.
The half-finished fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection, the described chromatographic condition of step (2) and step (3) is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30～35 ℃ of column temperatures, detection wavelength is 260～270nm.
As preferred version, the half-finished fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection, the described chromatographic condition of step (2) and step (3) is: with DianmonsilC18 (5 μ m, 4.6 * 250mm) for mensuration chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 40 ℃ of column temperatures, detection wavelength is 254nm.Linear gradient elution mode is: the ratio of mobile phase A in the time of 0 minute: B is 85:15, and the ratio of mobile phase A in the time of 25 minutes: B is 33:67, and the ratio of mobile phase A in the time of 65 minutes: B is 0:100.
As preferred version, the half-finished fingerprint atlas detection method of above-described Chinese medicine " kuhuang " injection, the described chromatographic condition of step (2) and step (3) is: with DianmonsilC18 (5 μ m, 4.6 * 250mm) for mensuration chromatographic column, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.Linear gradient elution mode is: the ratio of mobile phase A in the time of 0 minute: B is 98:2, the ratio of mobile phase A in the time of 20 minutes: B is 84:16, the ratio of mobile phase A in the time of 30 minutes: B is 80:20, and the ratio of mobile phase A in the time of 90 minutes: B is 40:60, and the ratio of mobile phase A in the time of 100 minutes: B is 0:100.
The application of Chinese medicine " kuhuang " injection fingerprint atlas detection method of the present invention in evaluating " kuhuang " injection end product quality.
The application of Chinese medicine " kuhuang " injection raw medicinal material fingerprint atlas detection method of the present invention in evaluating " kuhuang " injection raw medicinal material quality.
The application of the half-finished fingerprint atlas detection method of Chinese medicine " kuhuang " injection of the present invention in evaluating " kuhuang " injection semi-manufactured goods quality.
The present invention is by test sample finger-print and reference fingerprint machine similarity evaluation software (" the traditional Chinese medicine fingerprint area of computer aided similarity evaluation software 2004A version " of selecting Chinese Pharmacopoeia committee to recommend) calculating as calculated, obtain fingerprint similarity numerical value, according to similarity numerical value, be applied to " kuhuang " injection and raw medicinal material thereof, half-finished quality control.Fingerprint similarity should be greater than 0.90 for check quality control standard." kuhuang " injection provided by the invention and raw medicinal material thereof, half-finished fingerprint atlas detection method are for the production of laboratory quality controls, and products obtained therefrom has higher quality stability, can guarantee that product safety is effective.
Adopt the Chinese medicine " kuhuang " injection fingerprint atlas detection method that preferred version of the present invention provides 34 peaks to be detected altogether, wherein retention time is 83.4min ± 5%, and No. 32 peaks are Chrysophanol glucosylation compound, have obvious absorption peak, can be used as marker.
Wherein the relative retention time of typical detected peaks is respectively: 4.3 ± 5%(1 peak), 6.4 ± 5%(2 peak), 7.9 ± 5%(3 peak), 9.6 ± 5%(4 peak), 11.8 ± 5%(5 peak), 15.9 ± 5%(6 peak), 18.1 ± 5%(7 peak), 19.6 ± 5%(8 peak), 21.2 ± 5%(9 peak), 22.1 ± 5%(10 peak), 24.6 ± 5%(11 peak), 26min ± 5%(12 peak), 30min ± 5%(13 peak), 33.8 ± 5%(14 peak), 35.9 ± 5%(15 peak), 37.8 ± 5%(16 peak), 43.8 ± 5%(17 peak), 45.9 ± 5%(18 peak), 47.1 ± 5%(19 peak), 48.4 ± 5%(20 peak), 52.3 ± 5%(21 peak) and, 54.9 ± 5%(22 peak), 56min ± 5%(23 peak), 56.8 ± 5%(24 peak), 57.5 ± 5%(25 peak) 58.3 ± 5%(26 peak), 59.1 ± 5%(27 peak), 62.2 ± 5%(28 peak), 72.1 ± 5%(29 peak), 75.5 ± 5%(30 peak), 76.9 ± 5%(31 peak), 83.4min ± 5%(32 peak), 102min ± 5% (No. 33 peaks), 109min ± 5%(34 peak).
The fingerprint atlas detection method of " kuhuang " injection raw medicinal material provided by the invention, select the testing conditions identical with " kuhuang " injection finger print measuring method to carry out medicinal materials fingerprint investigation, be convenient to better investigate the correlativity of medicinal materials fingerprint and preparation finger, also be conducive to the relatively difference in each medicinal material place of production, for guaranteeing that the raw medicinal material of the safety and stability of " kuhuang " injection is originated, give security.
The top condition screening process of " kuhuang " injection provided by the invention and raw medicinal material thereof, semi-manufacture fingerprint atlas detection method:
1, instrument and reagent
Instrument: ShimadzuLC-10ATvp high performance liquid chromatograph, join double pump, degasser, automatic sampler, PDA detecting device, column oven; Waters2695 high performance liquid chromatograph, joins PDA and fluorescence detector; Agilent1100 high performance liquid chromatograph, joins degasser, quaternary gradient pump, automatic sampler, column oven, DAD detecting device, ELSD detecting device.
Reagent: acetonitrile, methyl alcohol are chromatographically pure (MERCK), it is pure that all the other reagent are analysis, and water is Milli-Q pure water.
Reference substance: matrine, Sophoridine, oxymatrine, indigo red, indigo, chlorogenic acid, aloe-emodin, Rhein, archen, Chrysophanol, Physcion (Nat'l Pharmaceutical & Biological Products Control Institute), 6,7-escoparone (Shenyang Pharmaceutical University provides), sophocarpine (Ningxia Hua Feng biological products company limited).
2, the screening of mobile phase kind
Because " kuhuang " injection active component is complicated, the present invention has screened methanol-water by great many of experiments, the mobile phases such as acetonitrile-water system, and owing to there being the acid ingredients such as chlorogenic acid, Rhein in " kuhuang " injection, at mobile phase, add acetic acid etc. to be conducive to peak shape and improve, therefore add a certain amount of acetic acid in methanol-water system, the last number at the demonstration peak by more different mobile phases, degree of separation and peak shape etc., last preferred mobile phase is: the HAc:H that (1) volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase; (2) Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase.
3, detect the selection of wavelength
Owing to containing anthraquinone in " kuhuang " injection, flavones and alkaloids reactive compound, as the Rhein in rheum officinale, Chrysophanol, aloe-emodin, archen, Physcion and glucoside thereof, chlorogenic acid in oriental wormwood, 2,6-escoparone, capillarisin, saikoside A in radix bupleuri, indigo, indigo red in folium isatidis etc.
According to the information theory of Shannon, certain signal, from information source is sent, is accepted the quantity of information that a side of this signal acquires and is
Φ
i＝log(1/p
i) (1)
Wherein, p
iprobability for signal appearance.
Obviously, the probability occurring when certain signal is larger, and the quantity of information of acquisition is less; Vice versa.If an information source is sent a large amount of signal (each signal is separate), its statistical mean information amount is that information entropy H is
Chromatogram under a certain wavelength is actually the concentration profile of each chemical composition, and its contained information entropy can be calculated by following formula:
H＝-∫p
xlogp
xdx (3)
Wherein, p
xit is the probability of x chemical composition in chromatogram.Here, p
xbe taken as m
x/ max[sum (m
x)].Wherein, m
xfor the chromatogram response of individual chemical composition, as peak area or peak height etc.Accordingly, the contained information entropy of the chromatogram under a certain wavelength is
H＝-∫m
x/max[sum(m
x)]logm
x/max[sum(m
x)]dx (4)
Calculate as stated above the information entropy of the " kuhuang " injection chromatogram acquiring under each wavelength.As shown in Figure 2, at 254nm and 262nm place, the contained quantity of information of its chromatogram reaches maximal value to result.Lower than the spectrogram of 250nm, noise ratio is larger, and baseline fluctuation is more severe, and a lot of composition does not respond or response is little; And higher than 280nm spectrogram, very multicomponent response is also obviously less than normal; By contrast spectrogram quantity of information and spectrogram peak shape, preferably 254nm and 262nm are as detecting wavelength.
4, the selection of eluent gradient
After having determined 2 kinds of mobile phase systems, mobile phase one: the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase; Mobile phase two: the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase.Owing to containing the various active compounds such as anthraquinone, flavones and alkaloids in " kuhuang " injection, peak shape is many and complicated, and the present invention has screened multiple different gradient elution program by great many of experiments, and 5 gradients (being linear gradient) condition is wherein:
（1）0min(10%B),50min(100%B),60min(100%B)
（2）0min（15％B），25min（67％B），65min（100％B）
（3）0min(2%B),20min(16%B),30min(20%B),60min(50%B),61min(100%B),70min(100%)
（4）0min(2%B),20min(16%B),30min(20%B),70min(100%B),100min(100%)
（5）0min(2%B),20min(16%B),30min(20%B),90min(60%B),100min(100%B),120min(100%)
Corresponding chromatogram is shown in Fig. 3.The chromatographic peak that considers separation case and provide is how many, the mobile phase two of chromatogram gradient (2) obtains for to(for) mobile phase one and gradient (5) is better separated and spectrum peak is more, can provide larger quantity of information, and it is short to have analysis time, analysis efficiency advantages of higher, therefore selected, using gradient (2) as the gradient elution chromatography (GEC) condition of mobile phase one, using the gradient elution chromatography (GEC) condition of gradient (5) as mobile phase two.
5, the selection of column temperature
The present invention analyzes " kuhuang " injection respectively under 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ of column temperatures, and Fig. 4 is shown in by gained finger-print.Experimental result shows, under 40 ℃ and 30 ℃ of column temperature conditions, chromatographic peak separation case is best, and post is pressed also lower, therefore, by 40 ℃ and 30 ℃, is optimum column temperature condition.
6, the selection of chromatographic column
In order to obtain more superior separating effect, the quality that more comprehensively reflects " kuhuang " injection, the present invention has screened different chromatographic columns by great many of experiments, the Jiangsu LichrospherC18 of Chinese nation post (5 μ m have been screened respectively, 4.6 * 250mm), WatersNova-pakC18 post (5 μ m, 4.6 * 150mm), AgilentZorbaxSB-C18 post (5 μ m, 4.6 * 250mm) and DikmaDiamonsilC18(5 μ m, 4.6 * 250mm), gained chromatogram is as Fig. 5.Experimental result shows, when selecting the DiamonsilC18 chromatographic column separating effect of enlightening horse to carry out.
7, different detecting devices are investigated
In order to investigate the impact of different detecting devices on finger-print, searching can provide the detection method compared with large information capacity, under above-mentioned chromatographic condition, adopts respectively UV-detector, fluorescence detector and DAD detector line to detect.Fluorescence detector parameter: excitation wavelength 254nm, emission wavelength 402nm; ELSD parameter: atomization gas is nitrogen, 2.0L/min, drift tube temperature is 100 ℃.Gained chromatogram is as Fig. 6.Experimental result shows, the chromatographic peak number that fluorescence and evaporative light-scattering detect gained " kuhuang " injection finger-print is obviously less than ultraviolet testing result, and the information providing is less, and therefore, the present invention selects UV-detector.
8, finger-print precision and stability
Get same " kuhuang " injection, in 24 hours, sample introduction is 6 times, the results are shown in Figure 7.Precision and good stability, take and analyze for the first time gained finger-print as contrast as seen from the figure, and the similarity result of calculation of analyzing gained finger-print for all the other 5 times is: 0.982,0.979,0.982,0.986,0.971, illustrate that consistance is fine.
9, reappearance
Get with 6 of a collection of " kuhuang " injections, according to aforementioned condition analysis, result is as Fig. 8.As take, wherein once analyze gained finger-print as contrast, analyze gained finger-print and its similarity for other 5 times and be respectively: 0.973,0.973,0.972,0.972,0.998; As contrasted with 6 average out to, the fingerprint similarity of analyzing for 6 times is respectively: 0.988,0.997,0.997,0.997,0.997,0.987.Show that the method has good reappearance.
Through above-mentioned research, the present invention has adopted optimum chromatogram condition one: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm, sample size: 10 μ l; Chromatographic condition two: chromatographic column filling agent is DianmonsilC18 chromatographic column, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.
Beneficial effect: Chinese medicine " kuhuang " injection fingerprint atlas detection method provided by the invention has the following advantages:
1, the present invention is according to active component contained in Chinese medicine Kuhuang injection, the structural property feature that comprises Anthraquinones, flavonoids and alkaloid compound, by great many of experiments, filter out best mobile phase composition, gradient elution program, flow velocity, detect wavelength, chromatographic column, the analysis conditions such as column temperature, through many experiments checking, show, Chinese medicine " kuhuang " injection fingerprint atlas detection method provided by the invention can quality comprehensive, objective, detecting and assessing " kuhuang " injection accurately be to guarantee that clinical efficacy is significant.
2, the fingerprint atlas detection method of each raw medicinal material of " kuhuang " injection provided by the invention, can detect the multiclass compounds such as Anthraquinones, flavonoids and alkaloid in " kuhuang " injection simultaneously, and can be used as " kuhuang " injection crude drug and half-finished fingerprint detection, there is purposes advantage more widely.And whole detection method, workable, there is sensitivity and precision is high, repeatability and good stability, can Accurate Determining and evaluate the quality of each raw medicinal material, for " kuhuang " injection guarantees the safety of raw medicinal material from source, effectively, holding gate of the quality monitoring has great importance.
Accompanying drawing explanation
Fig. 1 is " kuhuang " injection finger-print contrast collection of illustrative plates
Fig. 2 is the information entropy of " kuhuang " injection chromatogram under each wavelength
Fig. 3 is gained " kuhuang " injection chromatogram under best gradient condition
Fig. 4 is the impact of column temperature on " kuhuang " injection finger-print
Fig. 5 is the impacts of different chromatographic columns on " kuhuang " injection finger-print
Fig. 6 is the finger-print comparison of different detecting devices
Fig. 7 is that finger-print precision and stability test detect collection of illustrative plates
Fig. 8 is that reappearance test detects collection of illustrative plates
Fig. 9 is that the finger-print of " kuhuang " injection test sample detects collection of illustrative plates
Figure 10 is oriental wormwood medicinal material reference fingerprint
Figure 11 is rhubarb medicinal material reference fingerprint
Figure 12 is folium isatidis medicinal material reference fingerprint
Figure 13 is HERBA BUPLEURL medicinal material reference fingerprint
Figure 14 is Sophora flavescens reference fingerprint
Figure 15 is " kuhuang " injection semi-manufacture reference fingerprints
Figure 16 is " kuhuang " injection finger-print chromatographic peak numbering
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention, should understand these embodiment is only not used in and limits the scope of the invention for the present invention is described, after having read the present invention, those skilled in the art all fall within the application's claims limited range to the modification of the various equivalent form of values of the present invention.
Foundation and the application of embodiment 1 " kuhuang " injection reference fingerprint
1, the fingerprint atlas detection method of Chinese medicine " kuhuang " injection, it comprises the following steps:
(1) need testing solution preparation: get " kuhuang " injection as need testing solution;
(2) reference fingerprint of high effective liquid chromatography for measuring " kuhuang " injection: get 16 batches of " kuhuang " injection samples and record the finger-print of each batch of " kuhuang " injection by following chromatographic condition, as shown in Figure 1, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print in contrast;
34 total fingerprint peakses in finger-print, detected altogether, concrete retention time (min) is respectively:
4.3(1 number peak), 6.4(2 number peak), 7.9(3 number peak), 9.6(4 number peak), 11.8(5 peak), 15.9(6 peak), 18.1(7 peak), 19.6(8 peak), 21.2(9 peak), 22.1(10 peak), 24.6(11 peak), 26min(12 peak), 30min(13 peak), 33.8(14 peak), 35.9(15 peak), 37.8(16 peak), 43.8(17 peak), 45.9(18 peak), 47.1(19 peak), 48.4(20 peak), 52.3(21 peak), 54.9(22 peak), 56min(23 peak), 56.8(24 peak), 57.5(25 peak) 58.3(26 peak), 59.1(27 peak), 62.2(28 peak), 72.1(29 peak), 75.5(30 peak), 76.9(31 peak), 83.4(32 peak), 102 (No. 33 peaks), 109(34 peak).
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution (ratio of mobile phase A: B is 85:15 0 minute time, and the ratio of mobile phase A in the time of 25 minutes: B is 33:67, and the ratio of mobile phase A in the time of 65 minutes: B is 0:100), 40 ℃ of column temperatures; Detection wavelength is 254nm;
Or chromatographic condition is: with DianmonsilC18 (5 μ m, 4.6 * 250mm), be mensuration chromatographic column, and the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.Linear gradient elution mode is: the ratio of mobile phase A in the time of 0 minute: B is 98:2, the ratio of mobile phase A in the time of 20 minutes: B is 84:16, the ratio of mobile phase A in the time of 30 minutes: B is 80:20, and the ratio of mobile phase A in the time of 90 minutes: B is 40:60, and the ratio of mobile phase A in the time of 100 minutes: B is 0:100.
(3) high effective liquid chromatography for measuring need testing solution finger-print: get 24 batches of need testing solutions described in step 1, record the need testing solution finger-print of " kuhuang " injection by the identical chromatographic condition of step (2), as shown in Figure 9;
(4) get its similarity of similarity software evaluation for reference fingerprint that need testing solution finger-print that step (3) obtains and step (2) obtain, specific experiment result is as shown in table 1:
Table 1 " kuhuang " injection sample and similarity evaluation result
| Sequence number | Lot number | Similarity | Sequence number | | Similarity | |
| 1 | 1211291 | 0.948 | 13 | 1203011 | 0.976 | |
| 2 | 1211261 | 0.979 | 14 | 1202271 | 0.974 | |
| 3 | 1211061 | 0.964 | 15 | 1202211 | 0.976 | |
| 4 | 1210121 | 0.974 | 16 | 1202201 | 0.980 | |
| 5 | 1210081 | 0.973 | 17 | 1112071 | 0.960 | |
| 6 | 1206151 | 0.972 | 18 | 1112061 | 0.955 | |
| 7 | 1206081 | 0.955 | 19 | 1112051 | 0.964 | |
| 8 | 1205281 | 0.970 | 20 | 1111101 | 0.965 | |
| 9 | 1204271 | 0.958 | 21 | 1110311 | 0.953 | |
| 10 | 1204231 | 0.967 | 22 | 1105231 | 0.903 | |
| 11 | 1204161 | 0.981 | 23 | 1104121 | 0.907 | |
| 12 | 1203021 | 0.977 | 24 | 1103291 | 0.927 |
Result of calculation by above table 1 shows, each batch of " kuhuang " injection fingerprint similarity is higher, all be greater than 0.90, show that production technology is more stable, quality homogeneity is better, adopt fingerprint atlas detection method provided by the invention can objectively reflect the quality of " kuhuang " injection, for guaranteeing that clinical safety curative effect has great importance comprehensively.
Foundation and the application of the reference fingerprint of embodiment 2 " kuhuang " injection raw medicinal materials
1, the foundation of oriental wormwood medicinal material reference fingerprint and application
(1) need testing solution preparation: get 10 batches of oriental wormwood medicinal materials of various places, Linyi, Shandong, pulverize, get medicinal powder 4.0g, accurately weighed, add water 100ml, add hot reflux 1 hour, filter, get subsequent filtrate 50ml and be concentrated into about 10ml, add ethanol and make to reach 85% containing alcohol amount, placement is spent the night, and filters, the concentrated also water of filtrate is settled to 10ml, obtains need testing solution;
(2) reference fingerprint of high effective liquid chromatography for measuring raw material: get 10 batches of oriental wormwood raw medicinal material need testing solutions that step (1) prepares, by following chromatographic condition, record the finger-print of every batch of oriental wormwood raw medicinal material, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print in contrast;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm; (as shown in figure 10)
Or chromatographic condition is: with DianmonsilC18 (5 μ m, 4.6 * 250mm), be mensuration chromatographic column, and the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.Linear gradient elution mode is: the ratio of mobile phase A in the time of 0 minute: B is 98:2, the ratio of mobile phase A in the time of 20 minutes: B is 84:16, the ratio of mobile phase A in the time of 30 minutes: B is 80:20, and the ratio of mobile phase A in the time of 90 minutes: B is 40:60, and the ratio of mobile phase A in the time of 100 minutes: B is 0:100.
(3) high effective liquid chromatography for measuring oriental wormwood need testing solution finger-print: get the need testing solution of step (1) by the identical chromatographic condition sample introduction of step (2), record the finger-print of the need testing solution of " kuhuang " injection oriental wormwood raw medicinal material;
(4) get the oriental wormwood reference fingerprint similarity software evaluation that oriental wormwood need testing solution finger-print that step (3) obtains and step (2) obtain, the similarity of calculating respectively each batch of oriental wormwood medicinal materials fingerprint and reference fingerprint comparison, the results are shown in Table 2.Result shows that the fingerprint similarity of different batches oriental wormwood medicinal material is all greater than 0.90, show that oriental wormwood medicinal material reference fingerprint that the present invention sets up can be used for detecting and controlling the quality of the raw medicinal material oriental wormwood of " kuhuang " injection, for guaranteeing the safety of raw medicinal material, effectively, holding gate of the quality monitoring has great importance.
Table 2 oriental wormwood medicinal materials fingerprint similarity evaluation result
| Lot number | Similarity | Lot number | Similarity |
| 110509 | 0.989 | 110204 | 0.996 |
| 120503 | 0.981 | 110412 | 0.993 |
| 110608 | 0.996 | 120406 | 0.993 |
| 120110 | 0.986 | 121127 | 0.993 |
| 110830 | 0.987 | 121220 | 0.991 |
2, the foundation of rhubarb medicinal material reference fingerprint and application
(1) need testing solution preparation: get 10 batches of various places, the west of Gansu Province, Gansu rhubarb medicinal material, pulverize, get medicinal powder 4.0g, accurately weighed, add water 100ml, add hot reflux 1 hour, filter, get subsequent filtrate 50ml and be concentrated into about 10ml, add ethanol and make to reach 85% containing alcohol amount, placement is spent the night, and filters, the concentrated also water of filtrate is settled to 10ml, obtains need testing solution;
(2) reference fingerprint of high effective liquid chromatography for measuring raw material: get 10 batches of rheum officinale raw medicinal material need testing solutions that step (1) prepares, by following chromatographic condition, record the finger-print of every batch of rheum officinale raw medicinal material, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print (as shown in figure 11) in contrast;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm;
(3) high effective liquid chromatography for measuring rheum officinale need testing solution finger-print: get the rheum officinale need testing solution of step (1) by following chromatographic condition sample introduction, record the finger-print of the need testing solution of " kuhuang " injection rheum officinale raw medicinal material;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm;
(4) get the rheum officinale reference fingerprint similarity software evaluation that rheum officinale need testing solution finger-print that step (3) obtains and step (2) obtain, the similarity of calculating respectively each batch of rhubarb medicinal material finger-print and reference fingerprint comparison, the results are shown in Table 3.Result shows, the fingerprint similarity of the rhubarb medicinal material of 10 batches of different batches is all greater than 0.90, show that rhubarb medicinal material reference fingerprint that the present invention sets up can be used for detecting and controlling the quality of the raw medicinal material rheum officinale of " kuhuang " injection, for guaranteeing the safety of raw medicinal material, effectively, holding gate of the quality monitoring has great importance.
Table 3 rhubarb medicinal material fingerprint similarity evaluation result
| Lot number | Similarity | Lot number | Similarity |
| 130122 | 0.965 | 120828 | 0.930 |
| 130109 | 0.949 | 110830 | 0.921 |
| 110222 | 0.979 | 120110 | 0.961 |
| 111022 | 0.989 | 120406 | 0.920 |
| 110504 | 0.957 | 130507 | 0.923 |
3, the foundation of folium isatidis medicinal material reference fingerprint and application
(1) need testing solution preparation: get 10 batches of various places, Hui nationality folium isatidis medicinal material, pulverize, get medicinal powder 4.0g, accurately weighed, add water 100ml, add hot reflux 1 hour, filter, get subsequent filtrate 50ml and be concentrated into about 10ml, add ethanol and make to reach 85% containing alcohol amount, placement is spent the night, and filters, the concentrated also water of filtrate is settled to 10ml, obtains need testing solution;
(2) reference fingerprint of high effective liquid chromatography for measuring raw material: get 10 batches of folium isatidis raw medicinal material need testing solutions that step (1) prepares, by following chromatographic condition, record the finger-print of every batch of folium isatidis raw medicinal material, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print (as shown in figure 12) in contrast;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm;
(3) high effective liquid chromatography for measuring folium isatidis need testing solution finger-print: get the folium isatidis need testing solution of step (1) by following chromatographic condition sample introduction, record the finger-print of the need testing solution of " kuhuang " injection folium isatidis raw medicinal material;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm;
(4) get the folium isatidis reference fingerprint similarity software evaluation that folium isatidis need testing solution finger-print that step (3) obtains and step (2) obtain, the similarity of calculating respectively each batch of folium isatidis medicinal materials fingerprint and reference fingerprint comparison, the results are shown in Table 4.Experimental result shows, the fingerprint similarity of different batches folium isatidis medicinal material is all greater than 0.90.Showing that folium isatidis medicinal material reference fingerprint that the present invention sets up can be used for detecting and controlling the quality of the raw medicinal material folium isatidis of " kuhuang " injection, is the safety that guarantees raw medicinal material, effectively, holds gate of the quality monitoring and has great importance.
Table 4 folium isatidis medicinal materials fingerprint similarity evaluation result
| Lot number | Similarity | Lot number | Similarity |
| 130507 | 0.948 | 121220 | 0.944 |
| 100830 | 0.920 | 110526 | 0.901 |
| 121006 | 0.918 | 120313 | 0.963 |
| 110508 | 0.938 | 111212 | 0.917 |
| 110606 | 0.931 | 120110 | 0.919 |
| 110412 | 0.968 | 120214 | 0.914 |
4, the foundation of HERBA BUPLEURL medicinal material reference fingerprint and application
(1) need testing solution preparation: get 10 batches of HERBA BUPLEURL medicinal materials of From Quanjiao, Anhui Province various places, pulverize, get medicinal powder 4.0g, accurately weighed, add water 100ml, add hot reflux 2 hours, filter, get subsequent filtrate 50ml and be concentrated into about 10ml, add ethanol and make to reach 85% containing alcohol amount, placement is spent the night, and filters, the concentrated also water of filtrate is settled to 10ml, obtains need testing solution;
(2) reference fingerprint of high effective liquid chromatography for measuring raw material: get 10 batches of HERBA BUPLEURL raw medicinal material need testing solutions that step (1) prepares, by following chromatographic condition, record the finger-print of every batch of HERBA BUPLEURL raw medicinal material, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print (as shown in figure 13) in contrast;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm;
(3) high effective liquid chromatography for measuring HERBA BUPLEURL need testing solution finger-print: get the HERBA BUPLEURL need testing solution of step (1) by following chromatographic condition sample introduction, record the finger-print of the need testing solution of " kuhuang " injection HERBA BUPLEURL raw medicinal material;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm;
(4) get the HERBA BUPLEURL reference fingerprint similarity software evaluation that HERBA BUPLEURL need testing solution finger-print that step (3) obtains and step (2) obtain, the similarity of calculating respectively each batch of HERBA BUPLEURL medicinal materials fingerprint and reference fingerprint comparison, the results are shown in Table 5.Experimental result shows, the fingerprint similarity of different batches HERBA BUPLEURL medicinal material is all greater than 0.90, show that HERBA BUPLEURL medicinal material reference fingerprint that the present invention sets up can be used for detecting and controlling the quality of the raw medicinal material HERBA BUPLEURL of " kuhuang " injection, for guaranteeing the safety of raw medicinal material, effectively, holding gate of the quality monitoring has great importance.
Table 5 HERBA BUPLEURL medicinal materials fingerprint similarity evaluation result
| Lot number | Similarity | Lot number | Similarity |
| 130312 | 0.989 | 120327 | 0.998 |
| 110910 | 0.951 | 110822 | 0.992 |
| 111123 | 0.996 | 111024 | 0.996 |
| 110321 | 0.997 | 121012 | 0.981 |
| 110431 | 0.996 | 120126 | 0.993 |
5, the foundation of Sophora flavescens reference fingerprint and application
(1) need testing solution preparation: get 10 batches of various places, Qingzhou in Shandong province Sophora flavescens, pulverize, get medicinal powder 4.0g, accurately weighed, add water 100ml, add hot reflux 2 hours, filter, get subsequent filtrate 50ml and be concentrated into about 10ml, add ethanol and make to reach 85% containing alcohol amount, placement is spent the night, and filters, the concentrated also water of filtrate is settled to 10ml, obtains need testing solution;
(2) reference fingerprint of high effective liquid chromatography for measuring raw material: get 10 batches of kuh-seng raw medicinal material need testing solutions that step (1) prepares, by following chromatographic condition, record the finger-print of every batch of kuh-seng raw medicinal material, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print (as shown in figure 14) in contrast;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm;
(3) high effective liquid chromatography for measuring HERBA BUPLEURL need testing solution finger-print: get the kuh-seng need testing solution of step (1) by following chromatographic condition sample introduction, record the finger-print of the need testing solution of " kuhuang " injection kuh-seng raw medicinal material;
Chromatographic condition is: chromatographic column filling agent is DianmonsilC18 chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, 40 ℃ of column temperatures; Detection wavelength is 254nm;
(4) get the kuh-seng reference fingerprint similarity software evaluation that kuh-seng need testing solution finger-print that step (3) obtains and step (2) obtain, the similarity of calculating respectively each batch of Sophora flavescens finger-print and reference fingerprint comparison, the results are shown in Table 6.Result shows, the fingerprint similarity of different batches Sophora flavescens is all greater than 0.90, show that Sophora flavescens reference fingerprint that the present invention sets up can be used for detecting and controlling the quality of the raw medicinal material kuh-seng of " kuhuang " injection, for guaranteeing the safety of raw medicinal material, effectively, holding gate of the quality monitoring has great importance.
Table 6 Sophora flavescens fingerprint similarity evaluation result
| Lot number | Similarity | Lot number | Similarity |
| 130306 | 0.975 | 130702 | 0.988 |
| 110330 | 0.992 | 130408 | 0.992 |
| 120406 | 0.981 | 130221 | 0.984 |
| 111221 | 0.937 | 130619 | 0.991 |
| 130117 | 0.996 | 130515 | 0.995 |
Foundation and the application of embodiment 3 " kuhuang " injection semi-manufacture reference fingerprints
(1) need testing solution preparation: get " kuhuang " injection semi-manufacture as need testing solution." kuhuang " injection semi-manufacture preparation method: get by weight 250 parts of oriental wormwoods, 250 parts of HERBA BUPLEURLs, 80 parts of kuh-sengs, 100 parts of rheum officinales, 187 parts of folium isatidis, get oriental wormwood and HERBA BUPLEURL, use extraction by steam distillation volatile oil, collect gained volatile oil standby; The dregs of a decoction of oriental wormwood, HERBA BUPLEURL and kuh-seng, folium isatidis are merged to rear boiling 2 times, and each 1.5 hours, collecting decoction, was concentrated into relative density 1.10, added 85% ethanol precipitation 3 times, in 4 ℃ of refrigerations 48 hours, filtered filtrate for later use; Get rheum officinale boiling 2 times, each 1.5 hours, merge condensed water decocting liquid to relative density 1.05, add 85% ethanol precipitation 3 times, in 4 ℃ of refrigerations 48 hours, filter, above standby filtrate is merged, concentrated, add the volatile oil of steam distillation gained, mix, regulate pH to 7.0～7.5, obtain.
(2) the half-finished reference fingerprint of high effective liquid chromatography for measuring " kuhuang " injection: get 10 batches of " kuhuang " injection semi-manufacture samples by following chromatographic condition sample introduction, record each batch of half-finished finger-print of " kuhuang " injection, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print (as shown in figure 15) in contrast;
Chromatographic condition is: with DianmonsilC18 (5 μ m, 4.6 * 250mm), be mensuration chromatographic column, and the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, (ratio of mobile phase A: B is 98:2 to linear gradient elution 0 minute time, the ratio of mobile phase A in the time of 20 minutes: B is 84:16, the ratio of mobile phase A in the time of 30 minutes: B is 80:20, the ratio of mobile phase A in the time of 90 minutes: B is 40:60, the ratio of mobile phase A in the time of 100 minutes: B is 0:100), 30 ℃ of column temperatures, detection wavelength is 262nm.
(3) high effective liquid chromatography for measuring need testing solution finger-print: " kuhuang " injection semi-manufacture need testing solution to be measured, by the identical chromatographic condition sample introduction of step (2), is recorded to the finger-print of need testing solution;
(4) get its similarity of similarity software evaluation for reference fingerprint that need testing solution finger-print that step (3) obtains and step (2) obtain, specific experiment result is as shown in table 7:
Table 7 " kuhuang " injection semi-manufacture fingerprint similarity evaluation result
| Lot number | Similarity | Lot number | Similarity |
| 1303151 | 0.980 | 1302081 | 0.986 |
| 1303131 | 0.987 | 1310271 | 0.991 |
| 1302251 | 0.982 | 1310301 | 0.996 |
| 1303231 | 0.992 | 1212091 | 0.993 |
| 1304091 | 0.979 | 1212211 | 0.995 |
Data by table 7 show, the half-finished fingerprint similarity of different batches is very high, shows the stable processing technique of " kuhuang " injection, and product quality homogeneity is good.
Embodiment 4 raw medicinal materials, the correlativity of semi-manufacture and " kuhuang " injection preparation
Under the same terms described in embodiment 1, measure the finger-print of raw medicinal material, semi-manufacture and " kuhuang " injection preparation, the finger-print of " kuhuang " injection preparation and each raw medicinal material, semi-manufacture are compared one by one, to distinguish the correlativity of raw medicinal material, semi-manufacture and preparation.Each chromatographic peak numbering of " kuhuang " injection finger-print is shown in Figure 16, detects altogether 34 total fingerprint peakses.
1, rheum officinale and semi-manufacture, " kuhuang " injection preparation correlativity
Analyze under the same conditions the finger-print of rhubarb medicinal material, semi-manufacture and " kuhuang " injection test liquid.
Result shows, " kuhuang " injection and half-finished correlativity are fine, and peak shape is substantially consistent, and this is because the semi-manufacture of " kuhuang " injection are compared the difference in concentration just with preparation, and " kuhuang " injection is by semi-manufacture suitably after dilution and get final product, without other processing.
" kuhuang " injection is compared with rhubarb medicinal material, below main spectrum peak there is identical retention: 6.4min(2 peak), 7.9min(3 peak), 19.6min(8 peak), 21.2min(9 peak), 24.6min(11 peak), 26min(12 peak), 30min(13 peak), 35.9min(15 peak), 56min(23 peak), 56.8min(24 peak), 59.1min(27 peak), 75.5min(30 peak), 76.9min(31 peak), 83.4min(32 peak), 102min (No. 33 peaks), 109min(34 peak).
2, folium isatidis and semi-manufacture, " kuhuang " injection correlativity
Analyze under the same conditions the finger-print of folium isatidis medicinal material, semi-manufacture and " kuhuang " injection test liquid.
Result shows, folium isatidis is compared with " kuhuang " injection finger-print, and the chromatographic peak that retention time is consistent has: 1,2,3,4,5,6,7,9,10,16,17,24.
3, oriental wormwood and semi-manufacture, preparation correlativity
Analyze under the same conditions the finger-print of oriental wormwood medicinal material, semi-manufacture and " kuhuang " injection test liquid.
Result shows, the total peak of oriental wormwood and " kuhuang " injection is: 1,2,3,5,7,9,13,14,15,16,17,18,19,20,21,24,26,27.
4, kuh-seng and semi-manufacture, " kuhuang " injection correlativity
Analyze under the same conditions the finger-print of Sophora flavescens, semi-manufacture and " kuhuang " injection test liquid.
Result shows, the total peak of kuh-seng and " kuhuang " injection is: 3,7,8,14,21,24,28.
5, HERBA BUPLEURL and semi-manufacture, preparation correlativity
Analyze under the same conditions the finger-print of HERBA BUPLEURL medicinal material, semi-manufacture and " kuhuang " injection test liquid.
Result shows, the total peak of HERBA BUPLEURL and " kuhuang " injection is: 3,4,6,7,9,14,17,18,19,20,22,23,27.
Concrete comparative analysis result is as shown in table 8, and the corresponding relation of " kuhuang " injection finger-print and each medicinal material chromatographic peak is listed to 8.
Table 8 medicinal material and " kuhuang " injection finger-print correlativity
| Peak number | Retention time (min) | Rheum officinale | Kuh-seng | Folium isatidis | HERBA BUPLEURL | |
| 1 | 4.3 | √ | √ | |||
| 2 | 6.4 | √ | √ | √ | ||
| 3 | 7.9 | √ | √ | √ | √ | √ |
| 4 | 9.6 | √ | √ | |||
| 5 | 11.8 | √ | √ | |||
| 6 | 15.9 | √ | √ | |||
| 7 | 18.1 | √ | √ | √ | ||
| 8 | 19.6 | √ | √ | |||
| 9 | 21.2 | √ | √ | √ | √ | |
| 10 | 22.1 | √ | √ | |||
| 11 | 24.6 | √ | ||||
| 12 | 26.1 | √ | ||||
| 13 | 30.2 | √ | √ | |||
| 14 | 33.8 | √ | √ | √ | ||
| 15 | 35.9 | √ | √ | |||
| 16 | 37.8 | √ | √ | |||
| 17 | 43.8 | √ | √ | √ | ||
| 18 | 45.9 | √ | √ | |||
| 19 | 47.1 | √ | √ | |||
| 20 | 48.4 | √ | √ | |||
| 21 | 52.3 | √ | √ | |||
| 22 | 54.9 | √ | ||||
| 23 | 56.0 | √ | √ | |||
| 24 | 56.8 | √ | √ | √ | ||
| 25 | 57.5 | |||||
| 26 | 58.3 | √ | ||||
| 27 | 59.1 | √ | √ | √ | ||
| 28 | 62.2 | √ | ||||
| 29 | 72.1 | |||||
| 30 | 75.5 | √ | ||||
| 31 | 76.9 | √ | ||||
| 32 | 83.4 | √ | ||||
| 33 | 102.1 | √ | ||||
| 34 | 109.2 | √ |
From above-mentioned, relatively can find, " kuhuang " injection finger-print and each medicinal material all have certain correlativity, and wherein some spectrum peak is all relevant with a plurality of medicinal material, and this is because multi-flavor medicinal material all contains due to the total chemical compositions such as flavones.
By above experimental result, show, raw medicinal material provided by the invention, the fingerprint atlas detection method of semi-manufacture and " kuhuang " injection, can be objective, comprehensively, analyze targetedly and evaluate raw medicinal material, the quality of semi-manufacture intermediate and final " kuhuang " injection preparation, significant to guaranteeing clinical efficacy, fingerprint atlas detection method provided by the invention, can be simultaneously for analyzing raw medicinal material, semi-manufacture and " kuhuang " injection preparation, compared with the single analysis raw medicinal material of prior art, a kind of in semi-manufacture intermediate or final " kuhuang " injection, there is obvious technical progress.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (21)
1. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection, is characterized in that, comprises the following steps:
(1) need testing solution preparation: get " kuhuang " injection as need testing solution;
(2) reference fingerprint of high effective liquid chromatography for measuring " kuhuang " injection: get 10 batches of above " kuhuang " injection samples sample introduction under suitable chromatographic condition, record the finger-print of each batch of " kuhuang " injection, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print in contrast;
(3) high effective liquid chromatography for measuring need testing solution finger-print: " kuhuang " injection need testing solution to be measured, by the identical chromatographic condition sample introduction of step (2), is recorded to the finger-print of need testing solution;
(4) the reference fingerprint similarity software evaluation that need testing solution finger-print step (3) being obtained and step (2) obtain.
2. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection according to claim 1, is characterized in that, in step (2), chromatographic condition is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 35～40 ℃ of column temperatures, detection wavelength is 250～260nm.
3. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection according to claim 2, it is characterized in that, in step (2), chromatographic condition is: take specification as 5 μ m, the DianmonsilC18 of 4.6 * 250mm is mensuration chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 40 ℃ of column temperatures, detection wavelength is 254nm.
4. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection according to claim 3, it is characterized in that, described linear gradient elution mode is: the volume ratio of mobile phase A in the time of 0 minute: B is 85:15, the volume ratio of mobile phase A in the time of 25 minutes: B is 33:67, and the volume ratio of mobile phase A in the time of 65 minutes: B is 0:100.
5. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection according to claim 1, is characterized in that, in step (2), chromatographic condition is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the HAc:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30～35 ℃ of column temperatures, detection wavelength is 260～270nm.
6. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection according to claim 5, it is characterized in that, in step (2), chromatographic condition is: take specification as 5 μ m, the DianmonsilC18 of 4.6 * 250mm is mensuration chromatographic column, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.
7. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection according to claim 6, it is characterized in that, described linear gradient elution mode is: the volume ratio of mobile phase A in the time of 0 minute: B is 98:2, the volume ratio of mobile phase A in the time of 20 minutes: B is 84:16, the volume ratio of mobile phase A in the time of 30 minutes: B is 80:20, the volume ratio of mobile phase A in the time of 90 minutes: B is 40:60, and the volume ratio of mobile phase A in the time of 100 minutes: B is 0:100.
8. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection raw medicinal material, is characterized in that, comprises the following steps:
(1) need testing solution preparation: get " kuhuang " injection raw medicinal material, obtain after treatment the need testing solution of raw medicinal material;
(2) reference fingerprint of high effective liquid chromatography for measuring raw material: get respectively " kuhuang " injection raw medicinal material respectively more than 10 batches, disposal route by step (1) makes need testing solution, under suitable chromatographic condition, record the finger-print of every batch of raw medicinal material, take these finger-prints as basis, with similarity software, calculate and obtain common pattern as raw medicinal material reference fingerprint;
(3) high effective liquid chromatography for measuring need testing solution finger-print: get the need testing solution of step (1) by the identical chromatographic condition sample introduction of step (2), record the finger-print of the need testing solution of " kuhuang " injection raw medicinal material;
(4) the reference fingerprint similarity software evaluation that need testing solution finger-print step (3) being obtained and step (2) obtain.
9. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection raw medicinal material according to claim 8, is characterized in that, the described raw medicinal material disposal route of step (1) is: by pulverizing medicinal materials, get medicinal powder 4.0g, accurately weighed, add water 100ml, add hot reflux 1～2 hour, filter, get subsequent filtrate 50ml and be concentrated into about 10ml, add ethanol and make to reach 85% containing alcohol amount, placement is spent the night, filter, the concentrated also water of filtrate is settled to 10ml.
10. the fingerprint atlas detection method of Chinese medicine " kuhuang " injection raw medicinal material according to claim 8, is characterized in that, the chromatographic condition in step (2): chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 35～40 ℃ of column temperatures, detection wavelength is 250～260nm.
The fingerprint atlas detection method of 11. Chinese medicine " kuhuang " injection raw medicinal materials according to claim 10, it is characterized in that, in step (2), chromatographic condition is: take specification as 5 μ m, the DianmonsilC18 of 4.6 * 250mm is mensuration chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 40 ℃ of column temperatures, detection wavelength is 254nm.
The fingerprint atlas detection method of 12. Chinese medicine " kuhuang " injection raw medicinal materials according to claim 11, it is characterized in that, described linear gradient elution mode is: the volume ratio of mobile phase A in the time of 0 minute: B is 85:15, the volume ratio of mobile phase A in the time of 25 minutes: B is 33:67, and the volume ratio of mobile phase A in the time of 65 minutes: B is 0:100.
The fingerprint atlas detection method of 13. Chinese medicine " kuhuang " injection raw medicinal materials according to claim 8, is characterized in that, in step (2), chromatographic condition is: chromatographic column be take octadecylsilane chemically bonded silica as filling agent, the HAc:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30～35 ℃ of column temperatures, detection wavelength is 260～270nm.
The fingerprint atlas detection method of 14. Chinese medicine " kuhuang " injection raw medicinal materials according to claim 13, it is characterized in that, in step (2), chromatographic condition is: take specification as 5 μ m, the DianmonsilC18 of 4.6 * 250mm is mensuration chromatographic column, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.
The fingerprint atlas detection method of 15. Chinese medicine " kuhuang " injection raw medicinal materials according to claim 14, it is characterized in that, described linear gradient elution mode is: the volume ratio of mobile phase A in the time of 0 minute: B is 98:2, the volume ratio of mobile phase A in the time of 20 minutes: B is 84:16, the volume ratio of mobile phase A in the time of 30 minutes: B is 80:20, the volume ratio of mobile phase A in the time of 90 minutes: B is 40:60, and the volume ratio of mobile phase A in the time of 100 minutes: B is 0:100.
The fingerprint atlas detection method of 16. Chinese medicine " kuhuang " injection raw materials according to claim 8, is characterized in that, the raw medicinal material of " kuhuang " injection refers to rheum officinale, kuh-seng, oriental wormwood, HERBA BUPLEURL and folium isatidis medicinal material.
The half-finished fingerprint atlas detection method of 17. Chinese medicine " kuhuang " injection, is characterized in that, the method comprises the following steps:
(1) need testing solution preparation: get " kuhuang " injection semi-manufacture as need testing solution;
(2) the half-finished reference fingerprint of high effective liquid chromatography for measuring " kuhuang " injection: get 10 batches of above " kuhuang " injection semi-manufacture sample introduction under suitable chromatographic condition, record each batch of half-finished finger-print of " kuhuang " injection, take these finger-prints as basis, with similarity software, calculate and obtain common pattern finger-print in contrast;
(3) high effective liquid chromatography for measuring need testing solution finger-print: " kuhuang " injection semi-manufacture need testing solution to be measured, by the identical chromatographic condition sample introduction of step (2), is recorded to the finger-print of need testing solution;
(4) the reference fingerprint similarity software evaluation that need testing solution finger-print step (3) being obtained and step (2) obtain.
The half-finished fingerprint atlas detection method of 18. Chinese medicine " kuhuang " injection according to claim 17, it is characterized in that, chromatographic condition in step (2): take specification as 5 μ m, the DianmonsilC18 of 4.6 * 250mm is mensuration chromatographic column, the HAc:H that volume ratio is 1:100
2o is mobile phase A phase, the HAc:CH that volume ratio is 1:100
3oH is Mobile phase B phase, linear gradient elution, and 40 ℃ of column temperatures, detection wavelength is 254nm.
The half-finished fingerprint atlas detection method of 19. Chinese medicine " kuhuang " injection according to claim 18, it is characterized in that, described linear gradient elution mode is: the volume ratio of mobile phase A in the time of 0 minute: B is 85:15, the volume ratio of mobile phase A in the time of 25 minutes: B is 33:67, and the volume ratio of mobile phase A in the time of 65 minutes: B is 0:100.
The half-finished fingerprint atlas detection method of 20. Chinese medicine " kuhuang " injection according to claim 17, it is characterized in that, in step (2), chromatographic condition is: take specification as 5 μ m, the DianmonsilC18 of 4.6 * 250mm is mensuration chromatographic column, the Hac:H that volume ratio is 0.5:100
2o is mobile phase A phase, the HAc:H that volume ratio is 0.5:20:80
2o:CH
3cN is Mobile phase B phase, linear gradient elution, and 30 ℃ of column temperatures, detection wavelength is 262nm.
The half-finished fingerprint atlas detection method of 21. Chinese medicine " kuhuang " injection according to claim 20, it is characterized in that, described linear gradient elution mode is: the volume ratio of mobile phase A in the time of 0 minute: B is 98:2, the volume ratio of mobile phase A in the time of 20 minutes: B is 84:16, the volume ratio of mobile phase A in the time of 30 minutes: B is 80:20, the volume ratio of mobile phase A in the time of 90 minutes: B is 40:60, and the volume ratio of mobile phase A in the time of 100 minutes: B is 0:100.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310692364.6A CN103616459A (en) | 2013-12-17 | 2013-12-17 | Finger-print detection method of traditional Chinese medicine Kuhuang injection, and raw medicinal materials and semi-finished product thereof as well as application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310692364.6A CN103616459A (en) | 2013-12-17 | 2013-12-17 | Finger-print detection method of traditional Chinese medicine Kuhuang injection, and raw medicinal materials and semi-finished product thereof as well as application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103616459A true CN103616459A (en) | 2014-03-05 |
Family
ID=50167165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310692364.6A Pending CN103616459A (en) | 2013-12-17 | 2013-12-17 | Finger-print detection method of traditional Chinese medicine Kuhuang injection, and raw medicinal materials and semi-finished product thereof as well as application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103616459A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115015401A (en) * | 2022-02-16 | 2022-09-06 | 广州医科大学附属第五医院 | Fingerprint detection method of artemisia capillaris decoction compound preparation |
-
2013
- 2013-12-17 CN CN201310692364.6A patent/CN103616459A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 刘建华等: "RP-HPLC同时测定中药苦黄注射剂中4种生物碱的含量", 《云南中医中药杂志》 * |
| 吴永江: "苦黄注射液及若干药材质量分析方法研究", 《中国优秀博硕士学位论文全文数据库（博士）医药卫生科技辑》 * |
| 王恒斌等: "苦黄口服液质量标准研究", 《中国现代中药》 * |
| 顾巍华等: "HPLC测定苦黄注射液中大黄素、大黄酚的含量", 《中成药》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115015401A (en) * | 2022-02-16 | 2022-09-06 | 广州医科大学附属第五医院 | Fingerprint detection method of artemisia capillaris decoction compound preparation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104569252B (en) | A kind of method for building up of the finger printing of Chinese medicine composition | |
| CN111487344B (en) | Method for detecting fingerprint spectrum of motherwort particles | |
| CN105675744A (en) | Method for detecting fingerprint spectrum of cyclocarya paliurus | |
| CN102998375B (en) | A kind of method simultaneously detecting forulic acid and paeoniflorin content in blood-nourishing and brain-refreshing granules | |
| CN102520103A (en) | Contrast Chinese patent medicine extract of pills of six ingredients with rehmannia, and preparation method and application thereof | |
| CN102539607B (en) | Detection method for Traditional Chinese medicine fingerprint spectrum of Qishe pellet preparation | |
| CN110441413B (en) | Construction method and detection method of HPLC fingerprint of Qianbai rhinitis tablets | |
| CN107643343B (en) | HPLC fingerprint spectrum determination method of Yunv Jian standard soup | |
| CN109709222B (en) | Component detection method of Ganmaoling and compound Ganmaoling | |
| CN102879516A (en) | Method for identifying Buyang Huanwu soup and measuring content of Buyang Huanwu soup | |
| CN113189248B (en) | HPLC fingerprint construction and detection method of Yinhua Miyanling tablets | |
| CN108445125B (en) | HPLC fingerprint detection method of pain-relieving and illness-eliminating capsules | |
| CN103604878B (en) | A kind of finger-print acquisition methods of antiageing tablet, standard finger-print and application | |
| CN114942291B (en) | Quality detection method of yang-suppressing yin-nourishing particles | |
| CN103969356B (en) | A kind of discrimination method of the finger printing of red rooted salvia | |
| CN103616459A (en) | Finger-print detection method of traditional Chinese medicine Kuhuang injection, and raw medicinal materials and semi-finished product thereof as well as application thereof | |
| CN101926889A (en) | Method for detecting white paeony root-medlar particles | |
| CN102735766A (en) | Establishment method for polygonum perfoliatum medicinal material finger print, and standard finger print thereof | |
| CN103837619B (en) | Fingerprint detection method for traditional Chinese medicinal Ganyanlin injection and raw medicinal material thereof | |
| CN107607665A (en) | One seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control | |
| CN109781884B (en) | Establishing method of Qianliexin capsule fingerprint and fingerprint thereof | |
| CN106770785A (en) | A kind of method of the finger-print for setting up madder rattan medicinal material | |
| CN110308213B (en) | Method for constructing fingerprint spectrum of kidney-nourishing and fetus-growing pill and application of fingerprint spectrum in quality detection | |
| CN106290677A (en) | The method for building up of FENGLIAOXING FENGSHI DIEDA YAOJIU finger printing and finger printing thereof | |
| CN102590423A (en) | Fingerprint spectrum determination method of ligusticum wallichii tea-blending granular preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140305 |