CN104833733A - Finger-print spectrum mutual mode construction method and quality detection method of saussurea involucrate cell-disruption decoction pieces - Google Patents
Finger-print spectrum mutual mode construction method and quality detection method of saussurea involucrate cell-disruption decoction pieces Download PDFInfo
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Abstract
The invention relates to a finger-print spectrum mutual mode construction method and a quality detection method of saussurea involucrate cell-disruption decoction pieces. In the method, with rutin as a contrast, mutual mode standard spectrums and determination indexes are established through HPLC analysis to more than 10 batches of the samples under following chromatographic conditions: column temperature: 40 DEG C; wavelength: 285 nm; mobile phase A: methanol; mobile phase B: an 0.5% glacial acetic acid solution; elution gradient: 0 min-60 min; change of the mobile phase A: 20-65%; and sample size: 10 [mu]l. The spectrums of the samples to be detected under the same chromatographic condition are compared with the mutual mode standard spectrums to detect the quality of the samples to be detected. The invention firstly discloses the HPLC finger-print spectrum and the quality detection method aiming to the saussurea involucrate cell-disruption decoction pieces. The spectrums comprehensively contain the spectrum information of main active components of the saussurea involucrate cell-disruption decoction pieces. The method is strong in specificity, is quick and accurate in detection, and can effectively control the total quality of medicines, cell-disruption powders and cell-disruption decoction pieces.
Description
Technical field
The present invention relates to the quality determining method of the prepared slices of Chinese crude drugs, especially the finger-print common pattern of Herba Saussureae Involueratae broken wall medicine materical crude slice builds and quality determining method.
Background technology
Herba Saussureae Involueratae has another name called Saussurea involucrata, snow lotus of big bud flower, is the dry aerial parts of composite family phoenix hair Dendranthema Herba Saussureae Involueratae Saussureainvolucratea (Kar.etKir..) Sch.Bip..Main containing flavonoids, alkaloids, Phenylpropanoid Glycosides class, sesquiterpenoids, volatile oil and polysaccharide composition.Wherein, flavonoids mainly comprises rutin, Quercetin-3-O-α-L rhamnoside, acacetin, dinatin, Quercetin and apiolin etc.; Phenylpropanoid Glycosides class mainly comprises Syringin, chlorogenic acid and some coumarinoids; Alkaloids mainly comprises Involucratine; Sesquiterpenoids composition is mainly Involucratolactone constituents.
Li Yan etc. adopt HPLC-DAD method to measure the content of Syringin in Herba Saussureae Involueratae.Chromatographic condition is: Agilent Zorbax SB-C18 post (250mm × 4.6mm, 5 μm), and column temperature is 25 DEG C, and mobile phase is acetonitrile-water (10:90), flow velocity 1ml/min, and determined wavelength is 220nm.Yi Huangs etc. adopt HPLC method to measure the content of different parts rutin in Herba Saussureae Involueratae.Chromatographic condition is: CLC-ODS post (250mm × 4.6mm, 5 μm), column temperature is 35 DEG C, flow velocity 1ml/min, and mobile phase is 0.5% formic acid-acetonihile gradient elution.Lv Rui phoenix etc. for contrast, adopt HPLC method to measure 10 batches of Different sources Herba Saussureae Involueratae chromatograms with rutin, Syringin, chlorogenic acid.Chromatographic condition is: YMC-Pack ODS-A chromatographic column (250mm × 4.6mm, 5 μm), acetonitrile-1% acetate gradient wash-out (0 ~ 30 ~ 60min, acetonitrile is 10% ~ 20% ~ 20%), flow velocity 0.8ml/min, column temperature 30 DEG C, determined wavelength 270nm, result indicates 12 characteristic fingerprint peaks, and sample similarity is all more than 0.9.But when adopting said method to analyze Herba Saussureae Involueratae broken wall medicine materical crude slice, find that said method is all not exclusively applicable to the fingerprint map analyzing of Herba Saussureae Involueratae broken wall medicine materical crude slice, the characteristic peak in Herba Saussureae Involueratae broken wall medicine materical crude slice cannot be separated.
Chinese medicine wall cell disruption medicine materical crude slice utilizes modern superfine communication technique traditional medicine materical crude slice to be carried out breaking cell wall to be ground into the subparticle that size-grade distribution D90 is less than 45 μm, then make 30 ~ 100 object particles.Have color and luster relative to traditional medicine materical crude slice consistent, quality is homogeneous, the feature of good stability, is the innovative prepared slices of Chinese crude drugs.Because Chinese medicine wall cell disruption medicine materical crude slice does not have a morphological feature of traditional Chinese medicine medicine materical crude slice, the dissolution rate of the chemical composition such as effective constituent or index components can be brought after broken wall to change, make the discriminating of traditional medicine materical crude slice, quality determining method can not be completely applicable.Therefore, specificity must be set up for Chinese medicine wall cell disruption medicine materical crude slice strong, comprehensively the detection method of reflection Chinese medicine wall cell disruption prepared slice quality situation.And set up a new quality determining method not a duck soup, the such as selection of testing conditions, reference substance and the preparation method etc. of test sample all need research consider and verify.Although there is the detection method relating to traditional Chinese medicine fingerprint in prior art, but the method that there is no for the special Chinese medicine wall cell disruption medicine materical crude slice of form, and there is specificity, stability, reappearance and precision difference defect, can not the defect of quality condition of complete reaction medicine materical crude slice.
Summary of the invention
For overcoming the above problems the finger-print common pattern construction method that the invention provides a kind of Herba Saussureae Involueratae broken wall medicine materical crude slice, on this basis, further its quality determining method is disclosed.
Finger-print common pattern construction method of the present invention comprises the following steps:
(1) preparation of need testing solution: get Herba Saussureae Involueratae broken wall medicine materical crude slice and be about 1.000g, add 70% methyl alcohol 50ml, heating water bath refluxing extraction 45 minutes, supplies the weight of less loss with 70% methyl alcohol, filters, gets subsequent filtrate, to obtain final product;
(2) reference substance preparation: get control substance of Rutin, adds 70% methyl alcohol dissolving and is mixed with the reference substance solution that concentration is 0.0555mg/ml;
(3) chromatographic condition and system suitability: the chromatographic column taking octadecylsilane chemically bonded silica as filling agent, take methyl alcohol as mobile phase A, with 0.5% glacial acetic acid solution for Mobile phase B, gradient elution: 0min-60min, mobile phase A change 20%-65%; Column temperature is 40 DEG C; Determined wavelength is 285nm; Flow velocity: 1.0mL/min; Sample size: 10ul;
(4) by above-mentioned need testing solution and reference substance preparation method and and chromatographic condition HPLC analysis is carried out to the Herba Saussureae Involueratae broken wall medicine materical crude slice sample of more than 10 batches, obtain corresponding finger-print; With rutin peak for reference peak, calculate relative retention time and the relative peak area of main chromatographic peak in each finger-print; Input fingerprint map analyzing software, carries out similarity-rough set to each batch sample finger-print, with average spectrogram for common pattern, calculates similarity, sets up Herba Saussureae Involueratae broken wall medicine materical crude slice finger-print common pattern standard diagram, comprise 12 characteristic peaks.
Quality determining method comprises the following steps:
(1) testing sample need testing solution and reference substance solution is prepared respectively by step (1) and step (2) in above-mentioned finger-print common pattern construction method, by the chromatographic condition of step (3), accurate absorption testing sample solution, reference substance solution 10ul, inject HPLC chromatograph, measure, record chromatogram.
(2) qualified index foundation and specify collection of illustrative plates, determination methods:
In test sample chromatogram, 12 characteristic peaks corresponding with Herba Saussureae Involueratae broken wall medicine materical crude slice HPLC finger-print common pattern standard diagram should be presented; Calculate by similarity evaluation, the similarity of test sample finger-print and reference fingerprint must not lower than 0.9; With rutin peak for contrast peak, all the other 11 common characteristic peak relative retention times are respectively: 0.248,0.313,0.426,0.884,0.908,0.933,0.974,1.111,1.841,1.901,2.054, each peak relative retention time RSD≤± 5%, for qualified.
The present invention is first for the HPLC finger-print common pattern standard diagram constructed by the control of Herba Saussureae Involueratae broken wall prepared slice quality and quality determining method.The method overcome the defect that prior art can not be suitable for the control of Herba Saussureae Involueratae broken wall prepared slice quality completely.This collection of illustrative plates covers the profile information of Herba Saussureae Involueratae broken wall medicine materical crude slice main active more comprehensively, and specificity is obviously better than existing similar fingerprint spectrum method.All legal requirements is reached through the stability of verification experimental verification the method, reappearance and precision, and simple to operate, detect quick and precisely; And effectively can control Herba Saussureae Involueratae medicinal material, broken wall powder, broken wall medicine materical crude slice total quality and differentiate adulterant.
Accompanying drawing explanation
Fig. 1 is the Comparative map of different Extraction solvent.
Fig. 2 is variable concentrations methyl alcohol extraction effect Comparative map.
Fig. 3 be major component chromatographic peak point out Content of Chlorogenic Acid reference substance and Herba Saussureae Involueratae broken wall medicine materical crude slice chromatogram.
Fig. 4 be major component chromatographic peak point out central Tianshan saussurea involucrata broken wall medicine materical crude slice and Syringin reference substance chromatogram.
Fig. 5 be major component chromatographic peak point out central Tianshan saussurea involucrata broken wall medicine materical crude slice and control substance of Rutin chromatogram.
Fig. 6 is that Herba Saussureae Involueratae broken wall medicine materical crude slice finger-print specificity investigates collection of illustrative plates.
Fig. 7 is that Herba Saussureae Involueratae broken wall medicine materical crude slice has finger-print (No. 8 peaks are rutin peak).
Fig. 8 is Herba Saussureae Involueratae crude drug-broken wall powder-broken wall medicine materical crude slice finished product correlation analysis spectrogram.
Embodiment
1 instrument and sample
1.1 instruments: Agilent 1200RRLC fast liquid chromatography instrument (is furnished with G1315C model DAD detecting device, the ELSD evaporative light detecting device of G4218A, G1312B binary pump, G1329B automatic sampler, G1322A degasser, G1316B column oven, Agilent Chemstation chromatographic work station;
1.2 reagents:
Reagent: methyl alcohol (Sweden import Oceanpak chromatographic solvent), acetonitrile, chromatographically pure (Anhui Shi Lian special solvent company limited), distilled water, phosphoric acid, methyl alcohol (analyzing pure).Rutin, Syringin, chlorogenic acid reference substance is purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
Test sample: Herba Saussureae Involueratae broken wall medicine materical crude slice, Herba Saussureae Involueratae broken wall powder, Herba Saussureae Involueratae crude drug (group provides by Zhong Shanzhongzhi medicine company)
2 experimental techniques
2.1 trial test
The condition of Herba Saussureae Involueratae broken wall medicine materical crude slice HPLC fingerprint map analyzing is tentatively set: precision takes Herba Saussureae Involueratae broken wall medicine materical crude slice sample (powder 20121104) 1.000g, put in tool plug conical flask, add 50mL methyl alcohol, ultrasonic extraction 45min, let cool, filter, filtrate water bath method, add methyl alcohol dissolve and be settled to 10ml, 0.22um filtering with microporous membrane obtains test sample; Draft 4 sample elution system (methanol-waters; Acetonitrile-water; Methanol-water-phosphoric acid; Acetonitrile-water-phosphoric acid) difference wash-out test sample, chromatographic column is Agilent ZORBAX SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature is 25 DEG C, DAD detecting device, flow velocity is 1.0mL/min, sample size is 10l, the effect of 4 elution systems is compared respectively under 210nm, 254nm, 280nm and 325nm wavelength, preliminary screening goes out a preferably elution requirement (gradient 1-22), as the original chromatographic condition that later stage Herba Saussureae Involueratae sample preparation methods is investigated and chromatographic condition is investigated.
2.2 sample extraction preparation method study tours
2.2.1 the investigation of Extraction solvent
Because the main active in Herba Saussureae Involueratae is flavones and glycosides, Phenylpropanoid Glycosides and alkaloid, the dissolubility of this few constituents in ethyl acetate, ethanol and methyl alcohol is better, so this investigates the efficiency of ethyl acetate, ethanol and methyl alcohol 3 kinds of solvent extraction Herba Saussureae Involueratae broken wall medicine materical crude slice chemical compositions.
Sample preparation: the Herba Saussureae Involueratae broken wall medicine materical crude slice sample after precision takes and sieves is about 1.000g, 3 parts, be placed in tool plug conical flask, add the 1. each 50ml of ethanol, 2. methyl alcohol, 3. ethyl acetate respectively, soak 30min, ultrasonic extraction 45min, let cool, filter, filtrate water bath method, add methyl alcohol dissolve and be settled to 10ml, namely 0.22um filtering with microporous membrane obtains test sample.
Chromatographic condition: the chromatographic condition adopting 2.1 lower trial tests to filter out analyzes 3 increment product, Agilent ZORBAX SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature is 30 DEG C, DAD detecting device, flow velocity is 1.0ml/min, and sample size is 10 μ l, and determined wavelength is 270nm, 280nm, 285nm and 325nm.Difference is extracted by gradient 1-22 com-parison and analysis different solvents.Result display (see Fig. 1): methyl alcohol extracts sample gained spectrogram and goes out that peak number order is many, peak is evenly distributed and response is high, is optimum extraction solvent.
2.2.2 the investigation of Extraction solvent concentration
Investigate 50% methyl alcohol, 70% methyl alcohol, methyl alcohol 3 kinds of variable concentrations Extraction solvent respectively to the impact of Herba Saussureae Involueratae broken wall medicine materical crude slice chemical composition extraction efficiency.
Sample preparation: precision takes 3 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice samples and is about 1.000g, put in tool plug conical flask, measure 50% methyl alcohol, 70% methyl alcohol and each 50ml of methyl alcohol respectively, weighed weight, soak 30min, ultrasonic extraction 45min, take out, supply weightlessness after cooling, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Chromatographic condition: Agilent ZORBAX SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature is 30 DEG C, DAD detecting device, and flow velocity is 1.0mL/min, and sample size is 10 μ L, and determined wavelength is 270nm, 280nm, 285nm and 325nm.Difference is extracted by gradient 1-22 com-parison and analysis variable concentrations methyl alcohol.Result shows: it is few that 50% methanolic extract goes out peak number, and 70% methyl alcohol is almost identical with pure methanolic extract, goes out peak number many, and 70% methanolic extract spectrogram response the highest, pure methanolic extract collection of illustrative plates peak is evenly distributed.Considering optimum extraction solvent is 70% methyl alcohol.The results are shown in Figure 2.
2.2.3 the investigation of infiltrating time
Accurately weighed 4 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice are about 1.000g, put in tool plug conical flask, add 50ml70% methyl alcohol respectively, weighed weight, leave standstill 0min, 30min, 60min respectively and spend the night, ultrasonic extraction 45min, take out, supply weightlessness after cooling, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Chromatographic condition: Agilent ZORBAX SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature is 30 DEG C, DAD detecting device, flow velocity is 1.0mL/min, and sample size is 10 μ L, and determined wavelength is 270nm, 280nm, 285nm and 325nm, mobile phase is the phosphoric acid solution of methyl alcohol-0.3%, analyzes by gradient 1-22.The situation of the main chromatographic peak of com-parison and analysis each infiltrating time gained.Result shows: the extraction sample under different time of repose, and peak number difference is little, the sample for infiltrating 60min that the main chromatographic peak area of gained spectrogram is relatively large.Shown in table 1.
Table 1 compares infiltrating time impact
2.2.4 the investigation of extracting method
The each about 1.00g of accurately weighed 2 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice samples, puts in tool plug conical flask, adds 70% methyl alcohol 50ml respectively, weighed weight, infiltrate 30min, adopt following 2 kinds of methods to extract sample respectively: 1. ultrasonic extraction 45min (power 400W*0.8, frequency 40kHz); 2. heating water bath refluxing extraction 45min.Take out, after cooling, 70% methyl alcohol supplies weightlessness, shakes up, and 0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Chromatographic condition: Agilent ZORBAX SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature is 30 DEG C, DAD detecting device, and flow velocity is 1.0mL/min, and sample size is 10 μ L, and determined wavelength is 270nm, 280nm, 285nm and 325nm.Mobile phase methanol-0.3% phosphoric acid water system, gradient 1-22 analyzes, relatively two kinds of Different Extraction Method differences, show that both go out peak number difference by collection of illustrative plates and integration data little, about but refluxing extraction sample main chromatographic peak response doubles than ultrasonic extraction, so select heating and refluxing extraction.
2.2.5 extract centrifugal treating is investigated
Sample preparation: accurately weighed 2 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice are about 1.000g, put in tool plug conical flask, add 70% methyl alcohol 50mL respectively, weighed weight, infiltrates 30min, water-bath refluxing extraction 45min, takes out, supplies weightlessness, shake up after cooling.A copy of it sample directly adopts 0.22 μm of miillpore filter to filter, and gets subsequent filtrate as test sample; Another part of centrifugal 10min of 4000r/min, 0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Chromatographic condition: Agilent ZORBAX SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature is 30 DEG C, DAD detecting device, and flow velocity is 1.0mL/min, and sample size is 10 μ L, and determined wavelength is 270nm, 285nm.Mobile phase is methyl alcohol-0.3% phosphoric acid water system, and gradient 1-22 analyzes, more centrifugal difference.Drawn by collection of illustrative plates and integrating peak areas data: the response of centrifugal rear main chromatographic peak and peak area are slightly than not centrifugal height, but it is similar to go out peak number, in view of the easy contaminated environment of methyl alcohol centrifugally operated, the not centrifugal preparation method of final employing.
2.2.6 back flow of sample extraction time is investigated
Sample preparation: accurately weighed 4 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice samples are about 1.000g, put in tool plug conical flask, add 70% methyl alcohol 50ml respectively, weighed weight, infiltrates 60min, respectively water-bath refluxing extraction 30,45,60,120min, take out, supply weightlessness after cooling, shake up.0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Chromatographic condition: Agilent ZORBAX SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature is 30 DEG C, DAD detecting device, and flow velocity is 1.0mL/min, and sample size is 10 μ L, and determined wavelength is 270nm, 285nm.Mobile phase methanol-0.3% phosphoric acid water system, analyze by gradient 1-22, comparing reflux extracting time affects Herba Saussureae Involueratae broken wall medicine materical crude slice.Drawn by collection of illustrative plates and integrating peak areas data, the number difference that the sample of each return time goes out peak is little, and the peak area that backflow 45min extracts each main chromatographic peak of sample is relatively the most desirable, so select to add hot reflux 45min.Result is as shown in table 2.
Table 2 compares return time impact
2.3 chromatographic conditions are investigated
2.3.1 the selection of determined wavelength
Full wavelength scanner is carried out to sample, from its 3D spectrogram, selects best detection wavelength.Wherein 285nm is relatively the most desirable: under this wavelength, analytic signal response is large, and maximum absorption wavelength, takes into account out peak number amount many, and signal response value is large, and peak is evenly distributed and baseline is steady, and degree of separation is better, and overall visual effect is better.
2.3.2 the selection of mobile phase
According to the simplest, optimum screening principle screening Herba Saussureae Involueratae broken wall medicine materical crude slice mobile phase elution system.Finally select the best elution system of Herba Saussureae Involueratae sample, determine mobile phase composition and optimize gradient elution program, through the trace analysis of overwriting, result shows: take methyl alcohol as mobile phase A, the elute effect being Mobile phase B with 0.5% glacial acetic acid solution is obviously better than other test group, determine to take methyl alcohol as mobile phase A, with 0.5% glacial acetic acid solution for Mobile phase B is for flow phase system.
Mobile phase 1: be mobile phase A with methyl alcohol take water as Mobile phase B;
Mobile phase 2: be mobile phase A with acetonitrile take water as Mobile phase B;
Mobile phase 3: take methyl alcohol as mobile phase A, with 0.3% phosphoric acid solution for Mobile phase B;
Mobile phase 4: take methyl alcohol as mobile phase A, with 0.6% glacial acetic acid solution for Mobile phase B;
Mobile phase 5: take methyl alcohol as mobile phase A, with 0.5% formic acid solution for Mobile phase B;
Mobile phase 6: take methyl alcohol as mobile phase A, with 0.2% glacial acetic acid solution for Mobile phase B;
Mobile phase 7: take methyl alcohol as mobile phase A, with 0.3% glacial acetic acid solution for Mobile phase B;
Mobile phase 8: take methyl alcohol as mobile phase A, with 0.4% glacial acetic acid solution for Mobile phase B;
Mobile phase 9: take methyl alcohol as mobile phase A, with 0.5% glacial acetic acid solution for Mobile phase B;
Mobile phase 10: take methyl alcohol as mobile phase A, with 0.8% glacial acetic acid solution for Mobile phase B;
2.3.3 the investigation of eluent gradient
Use the gradient of Herba Saussureae Involueratae broken wall medicine materical crude slice methanolic extract to mobile phase to investigate by the gradient in table 3, result shows: analyze sample required time by gradient 1-36 short, and degree of separation, peak shape and post effect is obviously better than other test group.
The each gradient table of table 3 mobile phase
2.3.4 the selection of chromatographic column
Sample preparation: accurately weighed 4 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice samples are about 1.000g, put in tool plug conical flask, add 50mL70% methyl alcohol respectively, weighed weight, leave standstill 60min, heating water bath refluxing extraction 45min, take out, supply weightlessness after cooling, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Chromatographic condition: Agilent ZORBAX-C18 5 μm of (50mm × 4.6mm, 5um), Agilent ZORBAX EclipseC18 (100mm × 4.6mm, 1.8um), enlightening horse Diamonsil C18 (250mm × 4.6mm, 5um), Waters Symmetry C18 (250mm × 4.6mm, 5um), Féraud door PhenomenexSynergi 4u Fusion-RP 80A (250mm × 4.6mm, 5um) 5 kinds of different chromatographic columns, column temperature is 30 DEG C, DAD detecting device, flow velocity is 1.0mL/min, sample size is 10 μ L, determined wavelength is 270nm, 280nm, 285nm and 325nm, mobile phase is the glacial acetic acid solution of methyl alcohol-0.2%, analyze by gradient 1-36, result is: Agilent ZORBAX-C18 5 μm of (50mm × 4.6mm, 5um) pillar effect is relatively best, next is enlightening horse Diamonsil C-18 (250mm × 4.6mm, 5um).
2.3.5 the selection of test sample sample size
Sample preparation: accurately weighed 4 parts of Herba Saussureae Involueratae sample (powder) about 1.000g, put in tool plug conical flask, add 50mL70% methyl alcohol respectively, weighed weight, leave standstill 60min, heating water bath refluxing extraction 45min, take out, supply weightlessness after cooling, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Chromatographic condition: Agilent ZORBAX-C18 5 μm of (50mm × 4.6mm, 5um) post, column temperature is 30 DEG C, DAD detecting device, flow velocity is 1.0mL/min, and sample size is 10 μ L, determined wavelength is 270nm, 280nm, 285nm and 325nm, test sample sample size is respectively the glacial acetic acid solution of 5ul, 10ul and 20ul mobile phase methanol-0.2%, and gradient 1-36 analyzes, and result shows: sample size is that 10ul is the most applicable.
2.3.6 the selection of flow velocity
Sample preparation: accurately weighed 4 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice samples are about 1.000g, put in tool plug conical flask, add 50mL70% methyl alcohol respectively, weighed weight, leave standstill 60min, heating water bath refluxing extraction 45min, take out, supply weightlessness after cooling, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Chromatographic condition: enlightening horse Diamonsil C-18 (250mm × 4.6mm, 5um) post, column temperature is 30 DEG C, flow velocity is set to 0.8 respectively, 1,1.2mL/min, sample size 10 μ L, DAD detecting device determined wavelength 270nm, 280nm, 285nm and 325nm, mobile phase is the glacial acetic acid solution of methyl alcohol-0.2%, analyze by gradient 1-36, result shows: flow velocity 1.0ml/min analytical effect is the most desirable.
2.3.7 the investigation of chromatographic column column temperature
Sample preparation: accurately weighed 4 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice samples are about 1.000g, put in tool plug conical flask, add 50mL70% methyl alcohol respectively, weighed weight, leave standstill 60min, heating water bath refluxing extraction 45min, take out, supply weightlessness after cooling, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate as need testing solution.
Chromatographic condition: Agilent ZORBAX-C18 5 μm of (50mm × 4.6mm, 5um) post, column temperature is respectively 20,25,30,35,40 DEG C, flow velocity is 1.0mL/min, and sample size is 10 μ L, and DAD detecting device determined wavelength is 270nm, 280nm, 285nm and 325nm, test sample sample size is 10ul, mobile phase is the glacial acetic acid solution of methyl alcohol-0.2%, analyzes by gradient 1-36, and result shows: the column temperature analytical effect of 40 DEG C is the most desirable.
2.3.8 the pointing out of major component chromatographic peak
Sample preparation: accurately weighed 4 parts of Herba Saussureae Involueratae broken wall medicine materical crude slice samples are about 1.000g, put in tool plug conical flask, add 50mL70% methyl alcohol respectively, weighed weight, leave standstill 60min, heating water bath refluxing extraction 45min, take out, supply weightlessness after cooling, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate as test sample.
Prepared by reference substance: accurately take chlorogenic acid reference substance, rutin, Syringin reference substance respectively in right amount, add methyl alcohol and dissolve and be diluted to Syringin 11.98ug/ml, chlorogenic acid 23.776ug/ml, rutin 0.0555mg/ml.
Chromatographic condition: Agilent ZORBAX-C18 5 μm of (50mm × 4.6mm, 5um) posts, column temperature is 40 DEG C, DAD detecting device, flow velocity is 1.0mL/min, and sample size is 10 μ L, determined wavelength is 285nm, and mobile phase is the glacial acetic acid solution of methyl alcohol-0.5%, and gradient 1-36 analyzes.Rutin, Syringin is analyzed respectively according to Herba Saussureae Involueratae sample HPLC fingerprint analysis method, the reference substances such as chlorogenic acid, and with the comparison of Herba Saussureae Involueratae sample finger-print (increasing blank reagent to contrast), determine the correspondence position of each reference substance peak in total finger-print by spectrum and chromatogram, corresponding reference substance peak is pointed out.Result as shown in Figure 3-Figure 5, determines rutin, Syringin, the position of chlorogenic acid reference substance peak in common pattern.Wherein, No. 2 peaks are chlorogenic acid peak; No. 8 peaks are rutin peak; Syringin peak is less, is between No. 2 peaks and No. 3 peaks, and the relative retention time at itself and rutin peak is: 0.356.
2.4 methodological study
Determine best chromatographic condition by the result of study under 2.3, and methodological study is carried out to the method.Investigation method is with reference to " technical manual (trying) of traditional Chinese medicine finger-print research ".
2.4.1 specificity
Whether the Herba Saussureae Involueratae sample finger-print investigating the method foundation can express the feature of this kind, i.e. uniqueness.Whether investigation blank solvent collection of illustrative plates (negative sample collection of illustrative plates) with or without chromatographic peak at corresponding retention time place, has noiseless, meets the requirements.The results are shown in Figure 6, can draw there is no chromatographic peak at corresponding retention time place by collection of illustrative plates, noiseless, meet specificity requirement.
2.4.2 instrument precision experiment
Get same batch sample, by test sample, preparation method is prepared, continuous sample introduction 6 times, and record finger-print, the similarity calculating 6 sample introduction gained finger-prints is all greater than 0.9996, illustrates that Herba Saussureae Involueratae broken wall medicine materical crude slice fingerprint spectrum method precision is good.
Table 4 Herba Saussureae Involueratae broken wall medicine materical crude slice finger-print precision is investigated
2.4.3 repeated experiment
Get same batch of Herba Saussureae Involueratae broken wall medicine materical crude slice sample, by the parallel preparation of the preparation method of need testing solution 6 parts of test samples, record finger-print, Data Management Analysis is carried out by middle intelligence fingerprint map analyzing software, 6 spectrogram similarities are all greater than 0.9998, illustrate that this Herba Saussureae Involueratae broken wall medicine materical crude slice fingerprint spectrum method is reproducible.
Table 5 Herba Saussureae Involueratae broken wall medicine materical crude slice repeatability is investigated
2.4.4 stability experiment
With a Herba Saussureae Involueratae need testing solution, respectively at 0,2,4, measure in 8,12,24,48h, in data importing, intelligence fingerprint map analyzing software carries out analytic statistics, show that 6 spectrogram similarities are greater than 0.9994, illustrates that Herba Saussureae Involueratae broken wall medicine materical crude slice sample is relatively stable in 48h.
Table 6 study on the stability similarity data
The foundation of the common pattern of 2.5 Herba Saussureae Involueratae finger-prints
According to the above-mentioned best sample preparation method that determines and chromatographic condition, HPLC analysis is carried out to more than 10 batches Herba Saussureae Involueratae broken wall medicine materical crude slice samples, obtain corresponding finger-print.Comparatively large with peak area in collection of illustrative plates, peak shape is better, and the peak that its retention time is moderate is with reference to peak, calculates relative retention time and the relative peak area of each chromatographic peak in each ingredients fingerprint.Utilize Zhong Zhi medicine company group fingerprint map analyzing software, similarity-rough set (setting up with reference to finger-print with average) is carried out to each batch sample finger-print, calculate similarity, and set up Herba Saussureae Involueratae finger-print common pattern (as shown in Figure 7), take rutin as contrast peak, calculate the relative retention time of total fingerprint peaks, result is as shown in following table 7-table 9.
Table 70 batches of Herba Saussureae Involueratae broken wall medicine materical crude slice similarity tables
Table 80 batches of Herba Saussureae Involueratae broken wall medicine materical crude slice integrating peak areas tables
Table 90 batches of Herba Saussureae Involueratae broken wall medicine materical crude slice fingerprint image relative retention times
(remarks: sample 1-10 lot number is respectively: 20140601,20140602,20121124,0213081401,2014120501,2014120502,2014120503,2014120801,2014120802,2014120803)
Learn that different batches Herba Saussureae Involueratae main chemical compositions is similar by upper 3 tables, but different batches Herba Saussureae Involueratae chemical composition content otherness is larger, Herba Saussureae Involueratae broken wall prepared slice quality stable uniform is controlled to entirety, Herba Saussureae Involueratae raw medicinal material source must be fixed, steady production technique, is controlled to quality stable uniform.
4.3 Herba Saussureae Involueratae raw medicinal materials-broken wall powder-broken wall medicine materical crude slice finished product correlation analysis
Being used as medicine without the full composition of interpolation is Chinese medicine wall cell disruption medicine materical crude slice characteristic, because broken wall medicine materical crude slice and traditional medicine materical crude slice there occurs huge change in proterties, we, by the finger-print between contrast raw medicinal material, intermediate maturity, finished product, follow the tracks of the change of broken wall medicine materical crude slice in preparation process.Analyze intermediate-broken wall powder and the broken wall medicine materical crude slice finished product of 6 batches of raw medicinal materials and correspondence thereof respectively, in importing, intelligence fingerprint map analyzing software carries out standardization, the results are shown in following table 10 and Fig. 8.Result shows: three's chemical composition is substantially identical, and broken wall medicine materical crude slice intermediate (broken wall powder) and broken wall medicine materical crude slice finished product spectrogram similarity are all greater than 0.99.
Table 10 Herba Saussureae Involueratae is former-in-finished product correlation analysis
Two, the quality testing of Herba Saussureae Involueratae broken wall medicine materical crude slice
Test sample: in extraction intelligence medicine company Herba Saussureae Involueratae broken wall powder and broken wall medicine materical crude slice totally 10 batches carry out quality testing.Other producer's Herba Saussureae Involueratae broken wall medicine materical crude slice of market outsourcing 5 batches carry out quality testing.
Detection method is as follows:
The preparation of need testing solution: get Herba Saussureae Involueratae broken wall medicine materical crude slice and be about 1.000g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50ml, weighed weight, heating water bath refluxing extraction 45 minutes, take out, let cool, more weighed weight, the weight of less loss is supplied with 70% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Reference substance is prepared: the accurate control substance of Rutin that claims is appropriate, adds 70% methyl alcohol dissolving and is mixed with the mixing reference substance solution that concentration is respectively 0.0555mg/ml.
Chromatographic condition and system suitability: the chromatographic column (4.6mm × 250mm, 5 μm) taking octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol as mobile phase A, with 0.5% glacial acetic acid solution for Mobile phase B, the regulation according to the form below carries out gradient elution; Column temperature is 40 DEG C; Determined wavelength is 285nm; Flow velocity: 1.0mL/min.
Measure: accurate absorption test sample, reference substance solution 10 μ l, injecting chromatograph, measures, to obtain final product.
The foundation of qualified index and specify collection of illustrative plates, determination methods: in test sample chromatogram, should present 12 characteristic peaks corresponding with Herba Saussureae Involueratae broken wall medicine materical crude slice HPLC finger-print common pattern.Calculate by similarity evaluation, the similarity of test sample finger-print and reference fingerprint must not lower than 0.9.With rutin peak for contrast peak, all the other 12 common characteristic peak relative retention times are respectively: 0.248,0.313,0.426,0.884,0.908,0.933,0.974,1.111,1.841,1.901,2.054, each peak relative retention time RSD≤± 5%.
Result: middle intelligence medicine company 10 batch sample all conforms to quality requirements, outsourcing sample wherein 4 batches conform to quality requirements, 1 batch is unacceptable product.
Claims (2)
1. the construction method of the HPLC finger-print common pattern of Herba Saussureae Involueratae broken wall medicine materical crude slice, is characterized in that, comprise the following steps:
(1) preparation of need testing solution: get Herba Saussureae Involueratae broken wall medicine materical crude slice and be about 1.000g, add 70% methyl alcohol 50ml, heating water bath refluxing extraction 45 minutes, supplies the weight of less loss with 70% methyl alcohol, filters, gets subsequent filtrate, to obtain final product;
(2) reference substance preparation: get control substance of Rutin, adds 70% methyl alcohol dissolving and is mixed with the mixing reference substance solution that concentration is respectively 0.0555mg/ml;
(3) chromatographic condition and system suitability: the chromatographic column taking octadecylsilane chemically bonded silica as filling agent, take methyl alcohol as mobile phase A, with 0.5% glacial acetic acid solution for Mobile phase B, gradient elution: 0min-60min, mobile phase A change 20%-65%; Column temperature is 40 DEG C; Determined wavelength is 285nm; Flow velocity: 1.0mL/min; Sample size: 10 μ l.
(4) by the preparation method of above-mentioned test sample, reference substance and chromatographic condition, HPLC analysis is carried out to the Herba Saussureae Involueratae broken wall medicine materical crude slice sample of more than 10 batches, obtain corresponding finger-print; With rutin peak for reference peak, calculate relative retention time and the relative peak area of main chromatographic peak in each finger-print; Input fingerprint map analyzing software, carries out similarity-rough set to each batch sample finger-print, with average spectrogram for common pattern, calculates similarity, sets up Herba Saussureae Involueratae broken wall medicine materical crude slice finger-print common pattern standard diagram, comprise 12 characteristic peaks.
2. a quality determining method for Herba Saussureae Involueratae broken wall medicine materical crude slice, is characterized in that, comprises the following steps:
(1) testing sample need testing solution and reference substance solution is prepared respectively by step (1) in construction method according to claim 1 and step (2), by the chromatographic condition of step (3), accurate absorption testing sample, reference substance solution 10ul, inject HPLC chromatograph, measure, record chromatogram, to obtain final product.
(2) qualified index foundation and specify collection of illustrative plates, determination methods
In test sample chromatogram, 12 characteristic peaks corresponding with Herba Saussureae Involueratae broken wall medicine materical crude slice HPLC finger-print common pattern standard diagram should be presented; Calculate by similarity evaluation, the similarity of test sample finger-print and reference fingerprint must not lower than 0.9; With rutin peak for contrast peak, all the other 11 common characteristic peak relative retention times are respectively: 0.248,0.313,0.426,0.884,0.908,0.933,0.974,1.111,1.841,1.901,2.054, each peak relative retention time RSD≤± 5%, for qualified.
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