CN100392399C - Method for checking quality of Chinese Jin wine by using fingerprint pattern technology - Google Patents
Method for checking quality of Chinese Jin wine by using fingerprint pattern technology Download PDFInfo
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- CN100392399C CN100392399C CNB2005101205796A CN200510120579A CN100392399C CN 100392399 C CN100392399 C CN 100392399C CN B2005101205796 A CNB2005101205796 A CN B2005101205796A CN 200510120579 A CN200510120579 A CN 200510120579A CN 100392399 C CN100392399 C CN 100392399C
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Abstract
The invention discloses a method for detecting the vinosity of Chinese Jin alcohol by the fingerprint graph spectrum technique, the method includes following steps: the first step, the comparison fingerprint graph spectrums of Chinese Jin alcohol are built; the second step, the common mode graph spectrums of the fingerprint graph spectrum of Chinese Jin alcohol are built; the third step, detect the sample; and the graph spectrum of sample is compared with the comparison fingerprint graph spectrums, and the estimating system of auxiliary similarity is input the computer and calculate the similarity to obtain the correlation coefficient and can make a detection solution; the characteristics of the method are those: (1) it can represent the quality of clove medicinal materials macroscopically; (2) it is definitude and intuitionistic, and it has good exclusive property, characteristic property, repeatability; and good operation; (3) it can used to survey the stability of manufacturing technique and the stability of product; (4) it can be used to identify the true or false of the Chinese Jin alcohol; (5) it provides the demonstration for the product quality of health care alcohol.
Description
(1) technical field:
The present invention relates to the method that a kind of Chinese medicine health-care vinosity amount detects, be specifically related to the Chinese strength wine method for quality of high performance liquid chromatogram-gas chromatography fingerprint pattern technology detection by the plurality of Chinese feedstock production.
(2) prior art: Chinese strength wine is the health care wine with tonifying Qi, kidney tonifying, nourishing yin effect that Hubei strength board company produces, and mainly is prepared from by several Chinese crude drugs such as the Radix Astragali, saline cistanche, thizoma curculiginis, barrenwort, matrimony vine, Chinese yam, Radix Angelicae Sinensis, cloves, Chinese cassia trees.Aspect production quality control, before 2000, the major control index is color, solid content, alcoholic strength, mouthfeel, after 2000, has increased qualitative or quantitative targets such as icariin, general flavone, total saponin, alkaloid on the original basis.But as a Chinese medicine compound prescription health care wine, its health function mechanism and function factor are very complicated, can not control product quality fully by controlling several indexs, and must be controlled its material group integral body.So except micro-analysis, also need effectively to characterize the inherent quality of product on the whole with certain macroanalysis method.Traditional Chinese medicine fingerprint means the Chinese crude drug in certain or certain place of production or Chinese patent drug after suitably handling, and adopts collection of illustrative plates such as the chromatogram that can indicate this Chinese crude drug or Chinese patent drug feature that certain analysis means obtains, spectrum.As the traditional Chinese medicine quality control method, in the ascendant to the research of finger-print both at home and abroad.In the world, collection of illustrative plates contains compounding substances group's method of quality control as Chinese patent drug, herb extracts etc., has become the world of medicine's common recognition.U.S. FDA (Food and Drug Administration) requires to work out the examination criteria of finger-print to the quality testing of autonomic drug, so at the CMC (Chemistry that declares IND (Investigational New Drug), Manufacture and Control) during data, the quality control of plant material (Botanical DrugSubstance) and autonomic drug product (Botanical Drug product) will be adopted finger-print.The European Community has also adopted fingerprint pattern technology to the quality control of herbal medicine.Japan fries in shallow oil juice to what the decoction of authentic medicinal herbs medicine materical crude slice prescription obtained as standard extract, the finger-print of standard extract is standard finger-print, with this raw material, prescription and the technology of producing are done strict control, make the finished product finger-print consistent with standard finger-print.In addition, France, Germany, Britain, India and WHO etc. adopt fingerprint pattern technology to carry out the quality assessment of autonomic drug (herbal medicine).Bureau of Drugs Supervision of country issued " technical requirement of the finger-print research of traditional Chinese medicine " in 2000, require traditional Chinese medicine is carried out finger-print Quality Control way.And other Chinese patent medicine preparations employing traditional Chinese medicine fingerprint Quality Control technology also are the inexorable trend of Chinese medicine development as quality inspection standard.Therefore, adopt finger-print Quality Control technology, provide quality goods or brilliant and aspect such as production run control will play a significant role at real and fake discrimination, quality control, the product of Chinese medicine and Related product.
(3) technology contents: purpose of the present invention is exactly to utilize the globality of traditional Chinese medicine fingerprint, characteristic and quantification property, propose a kind of can be on macroscopic view, detect Chinese strength wine method for quality on the whole.Simultaneously, also provide demonstration for China's health care wine industry production quality control.
The present invention includes following step:
The first step is set up Chinese strength wine reference fingerprint
1. the preparation of Chinese strength wine
Take off and state parts by weight of Chinese traditional medicine raw material Radix Astragali 10-15, saline cistanche 10-15, thizoma curculiginis 10-15, barrenwort 10-15, matrimony vine 10-15, Chinese yam 10-15, Radix Angelicae Sinensis 10-15, cloves 3-5, Chinese cassia tree 3-5 and filter with the bent Bai Jiu of the 30-50 ° of Xiao Seepage of 50 times of total medicinal material amounts and got percolate in 12-15 days, get percolate 70-75 part, it is an amount of to add 20 parts in 40% syrup, pure water 5-10 part, burnt sugar coloring respectively, allotment is filtered, and puts under the normal temperature ageing 3-6 month, filtering and impurity removing, the smart filter of filter membrane, can, promptly;
2. the preparation of Chinese strength wine liquid phase part reference substance solution
A. ether position
Get Chinese strength wine 100ml,, use water saturation extracted with diethyl ether 3 times with macroporous absorption chromatographic column desaccharification, each 20ml, combining extraction liquid, evaporate to dryness is settled to 2ml with tetrahydrofuran, membrane filtration, promptly;
B.5% the ethyl acetate extract of isoamylol
With ether position need testing solution with the ethyl acetate extraction of water saturated 5% isoamylol 3 times, each 20ml, combining extraction liquid, evaporate to dryness is settled to 2ml with tetrahydrofuran, membrane filtration, promptly;
3. the preparation of Chinese strength fume phase part reference substance solution
Directly get Chinese strength wine 5ml in 10mi headspace sampling bottle, seal, promptly;
4. the preparation of standard solution
A. liquid phase part
It is an amount of to get the forulic acid standard items, makes the solution of lmg/ml with 50% methyl alcohol; It is an amount of to get the icariin standard items, makes the solution of lmg/ml with 50% methyl alcohol;
B. gas phase part
Dip in glass capillary and to get Bronyl acetate, eugenol standard items in right amount in the headspace sampling bottle, sealing, promptly;
5. chromatographic condition
A. liquid phase part
Chromatographic column
The ether position: Hypersil C18 post,
The ethyl acetate extract of 5% isoamylol: Aichrombond AQ C18,
Moving phase
The ether position: acetonitrile-0.01% phosphate aqueous solution is the eluent gradient wash-out;
The ethyl acetate extract of 5% isoamylol: acetonitrile-0.01% phosphate aqueous solution is the eluent gradient wash-out;
Flow velocity: 0.8ml/ minute
Detect wavelength: 280nm
Column temperature: 30 ℃
B. gas phase part
Chromatographic column Agilent HP-5
Carrier gas is N2
Injector: G1888A head space automatic sampler;
6. reference substance detects
A. liquid phase part
Ether position: accurate respectively Chinese strength wine liquid phase part ether position reference substance solution and each 4 μ l of standard solution of drawing, inject liquid chromatograph, according to high effective liquid chromatography for measuring, promptly get Chinese strength wine ether position reference fingerprint and corresponding standard product finger-print;
The ethyl acetate extract of 5% isoamylol: accurate respectively ethyl acetate extract reference substance solution and each 4 μ l of standard solution that draw Chinese strength wine liquid phase part 5% isoamylol, inject liquid chromatograph, according to high effective liquid chromatography for measuring, the ethyl acetate extract that promptly gets 5% amylalcohol contrasts different finger-print and corresponding standard product finger-print;
B. gas phase part
Respectively gas phase part reference substance solution and standard solution are carried out assay determination by the head-space sampler auto injection, promptly get gas phase part reference fingerprint and corresponding standard product finger-print;
In the described Chinese strength wine reference fingerprint, the common characteristic peak is 10 in the reference fingerprint at liquid phase part ether position, wherein No. 7 peaks are forulic acid, the common characteristic peak is 9 in the ethyl acetate extract reference fingerprint of 5% amylalcohol, wherein No. 11 peaks are icariin, and are consistent with the standard items finger-print; The common characteristic peak is 8 in the described gas phase part reference fingerprint, and wherein No. 7 peaks are Bronyl acetate, and No. 8 peaks are eugenol, and are consistent with the standard items finger-print;
The concrete grammar of the ethyl acetate extract reference substance solution preparation of described Chinese strength wine liquid phase part ether position and 5% isoamylol is, get Chinese strength wine 100ml, 40 ℃ of water-bath rotary evaporations are done near, be splined on the macroporous absorption chromatographic column that AB-8 is housed (12mm * 300mm), water is eluted to the sugar-free reaction, (being that the Molish reaction is negative), back 80ml95% ethanol elution, collect eluent, evaporated under reduced pressure.Add 20ml water, ultrasonicly make dissolving, and be transferred in the separating funnel.Use water saturation extracted with diethyl ether 3 times earlier, each 20ml, combining extraction liquid, water bath method is settled to 2ml with tetrahydrofuran, shakes up, and with 0.45 μ filtering with microporous membrane, promptly gets ether position reference substance solution; The ethyl acetate extraction of 5% again that this solution with water is saturated isoamylol 3 times, each 20ml, combining extraction liquid, evaporated under reduced pressure is settled to 2ml with tetrahydrofuran, shakes up, with 0.45 μ filtering with microporous membrane, promptly get the ethyl acetate extract reference substance solution of 5% isoamylol;
Described liquid phase part ether position is the eluent gradient wash-out with acetonitrile-0.01% phosphate aqueous solution, its gradient is: when retention time is 0min, 45min, 80min, 90min, 105min, the volume ratio of acetonitrile and 0.01% phosphate aqueous solution was respectively 8: 92,30: 70,50: 50,80: 20,80: 20;
The ethyl acetate extract of 5% isoamylol: acetonitrile-0.01% phosphate aqueous solution is the eluent gradient wash-out, its gradient is: when retention time is 0min, 50min, 80min, 100min, 110min, the volume ratio of acetonitrile and 0.01% phosphate aqueous solution was respectively 15: 85,30: 70,55: 45,100: 0,100: 0;
Described computer aided similarity evaluation system means the computer aided similarity evaluation system by the modernization of cmm center exploitation of Hunan Central South University that pharmacopoeia commission is recommended;
The foundation of China's strength wine reference fingerprint makes the quality control of product that standard very clear and definite, directly perceived, easy operating arranged, and only testing result need be compared with it during use to get final product.
In second step, set up Chinese strength wine finger-print common pattern collection of illustrative plates
Prepare 10 batches Chinese strength wine and standard items, need testing solution by the preparation method of the preparation method of the preparation method of Chinese strength wine, Chinese strength wine liquid phase part and gas phase part reference substance solution, standard solution, and detect by same procedure one by one, obtain ethyl acetate extract finger-print, gas phase part finger-print and the corresponding standard product finger-print of the Chinese strength wine of each batch ether position finger-print, 5% isoamylol respectively; Import computer aided similarity evaluation system respectively and calculate, can draw the ethyl acetate extract of Chinese strength wine ether position, 5% isoamylol and the finger-print common pattern collection of illustrative plates of gas phase part;
The effect one of setting up the common pattern collection of illustrative plates is the related coefficient that testing result can be imported between COMPUTER CALCULATION test sample and the total mode chart spectrum, investigate the similarity between test sample and the total mode chart spectrum theoretically, thereby reach the purpose of control product quality; The 2nd, compare between convenient a plurality of batches the product, to investigate the stability of production technology, the stability of product is to guarantee the being product that the consumer provides high-quality.
In the 3rd step, test sample detects
1. the preparation of need testing solution
The ethyl acetate need testing solution and the gas phase part need testing solution that prepare liquid phase part ether position and 5% isoamylol with above-mentioned same procedure respectively;
2. prepare liquid phase part and gas phase part of standards product solution respectively with above-mentioned same procedure;
3. measure standard items and test sample to be measured with above-mentioned same procedure;
4. interpretation of result is judged
Should detect 10 characteristic peaks identical with reference fingerprint in the test sample finger-print of ether position, wherein No. 7 peaks are forulic acid, should be consistent with the standard items finger-print; Should detect 9 characteristic peaks identical in the ethyl acetate extract test sample finger-print of 5% isoamylol with reference fingerprint; Wherein No. 11 peaks are icariin, should be consistent with the standard items finger-print; The finger-print of two parts and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient should be greater than 0.9000;
Should detect 8 characteristic peaks identical in the gas phase part test sample finger-print with reference fingerprint; Wherein No. 7 peaks are Bronyl acetate, and No. 8 peaks are eugenol, should be consistent with the standard items finger-print; Its finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient should be greater than 0.9000;
Principle of the present invention is based on to contain in a large amount of involatile constituents and the part material in the Chinese strength wine and contains volatile ingredient, and involatile constituent has stronger absorption in the ultraviolet region, because Chinese strength wine is to be prepared from by the multidigit medicinal material, complex chemical composition, character according to its main chemical compositions, finished product is separated into the ethyl acetate extract of ether position and 5% isoamylol, adopts the liquid and gas chromatogram that involatile constituent and volatile ingredient are detected respectively; In research process, also respectively extracting method, detection method, chromatographic condition and system flexibility, accuracy, stability, reappearance etc. are investigated, according to the testing result of 10 batches of above pilot products, product reference fingerprint and common pattern collection of illustrative plates have been set up; And the related coefficient between definite test sample finger-print and the corresponding common pattern collection of illustrative plates is 0.9000; Therefore, can hold the quality situation of Chinese strength wine from the whole facial feature of chromatogram.
Be the partial results that this method is investigated below:
(1) reappearance test
Get same sample solution, press test sample preparation method and detection method operation 6 times, calculate similarity, the result shows, the liquid-phase fingerprint of the ethyl acetate extract of China strength wine ether position and 5% isoamylol and the similarity of 6 testing results of gas phase part finger-print are all greater than 0.9000, and the method reappearance is better;
(2) ten batch sample finger-print similarities are investigated
10 batches of Chinese strength wine finger-prints to random collecting carry out the similarity investigation, result of calculation shows, 10 batches similarity is all greater than 0.9000, prompting this method can not only be controlled the inherent quality of strength wine product on the whole, can also effectively monitor the stability of production technology and the stability of product;
(3) commercial like product relatively
With this method commercially available 8 kinds of like products are detected, compare each product fingerprint collection of illustrative plates, can significantly find out: the liquid-phase fingerprint of the ether position of these 8 kinds health care wine and the ethyl acetate extract of 5% isoamylol and the Chinese strength wine that our company produces all have very big difference, illustrate that detection method of the present invention has specificity and characteristic preferably; Can not only control product quality, can also effectively discern our company's product;
Four, description of drawings
Fig. 1 is a Chinese strength wine ether position reference fingerprint
Fig. 2 is the ethyl acetate extract reference fingerprint of Chinese strength wine 5% isoamylol
Fig. 3 is Chinese strength wine finished product gas phase part reference fingerprint
Fig. 4 is a Chinese strength wine ether position common pattern collection of illustrative plates
Fig. 5 is the ethyl acetate extract common pattern collection of illustrative plates of Chinese strength wine 5% isoamylol
Fig. 6 is Chinese strength fume phase part common pattern collection of illustrative plates
Fig. 7 is that lot number is 20041220 Chinese strength wine ether position finger-prints
Fig. 8 is that lot number is the ethyl acetate extract finger-print of 20041220 Chinese strength wine 5% isoamylol
Fig. 9 is that lot number is 20041220 Chinese strength fume phase part finger-prints
Figure 10 is that lot number is 20050425 Chinese strength wine ether position finger-prints
Figure 11 is that lot number is the ethyl acetate extract finger-print of 20050425 Chinese strength wine 5% isoamylol
Figure 12 is that lot number is 20050425 Chinese strength fume phase part finger-prints
Figure 13 is that lot number is 20050723 Chinese strength wine ether position finger-prints
Figure 14 is that lot number is the ethyl acetate extract finger-print of 20050723 Chinese strength wine 5% isoamylol
Figure 15 is that lot number is 20050723 Chinese strength fume phase part finger-prints
Figure 16 is 20041220,20050425,20,050,723 3 lot number China strength wine ether position finger-print similarity evaluations figure as a result
Figure 17 is the ethyl acetate extract finger-print similarity evaluation figure as a result of 20041220,20050425,20,050,723 3 lot numbers China strength wine 5% isoamylol
Figure 18 is 20041220,20050425,20,050,723 3 lot number China strength fume phase part finger-print similarity evaluations figure as a result
Figure 19 is forulic acid standard items finger-prints
Figure 20 is icariin standard items finger-prints
Figure 21 is Bronyl acetate standard items finger-prints
Figure 22 is eugenol standard items finger-prints
(5) embodiment:
Embodiment 1 lot number is the quality testing of 20041220 Chinese strength wine
1, sample source strength board company warehouse
2, the sample lot number 20041220
3, the preparation of standard solution
A. liquid phase part
It is an amount of to get the forulic acid standard items, makes the solution of 1mg/ml with 50% methyl alcohol; It is an amount of to get the icariin standard items, makes the solution of 1mg/ml with 50% methyl alcohol;
B. gas phase part
Dip in glass capillary and to get Bronyl acetate, eugenol standard items in right amount in the headspace sampling bottle, sealing, promptly;
4, the preparation of need testing solution
Get lot number and be 20041220 Chinese strength wine 100ml, 40 ℃ of water-bath rotary evaporations are done near, be splined on the macroporous absorption chromatographic column that AB-8 is housed (12mm * 300mm), water is eluted to the sugar-free reaction, (being that the Molish reaction is negative), eluent is collected, evaporated under reduced pressure with 80ml 95% ethanol elution in the back.Add 20ml water, ultrasonicly make dissolving, and be transferred in the separating funnel.Use water saturation extracted with diethyl ether 3 times earlier, each 20ml, combining extraction liquid, water bath method is settled to 2ml with tetrahydrofuran, shakes up, and with 0.45 μ filtering with microporous membrane, promptly gets ether position need testing solution; The ethyl acetate extraction of 5% again that this solution with water is saturated isoamylol 3 times, each 20ml, combining extraction liquid, evaporated under reduced pressure is settled to 2ml with tetrahydrofuran, shakes up, with 0.45 μ filtering with microporous membrane, promptly get the ethyl acetate extract need testing solution of 5% isoamylol;
5, key instrument and reagent
A. instrument
Chromatograph Varian Associates, Inc. (US) 611 Hansen Way, Palo Alto, California 94303, U.S.A. instrument (230 pumps, 330 detecting device 7725i manual injector AT-830 column ovens) and Agilent6890
Japan EYELA1000 type Rotary Evaporators
B. reagent
It is pure that analysis is produced in ether Tianjin
It is pure that analysis is produced in isoamylol Tianjin
It is pure that analysis is produced in ethyl acetate Beijing
Acetonitrile USAJ.T.Baker chromatographically pure
It is pure that analysis is produced in phosphoric acid Beijing
Water Robust pure water single flash
The N2 high pure nitrogen, purity 99.999%
China strength wine sample Hubei strength board company produces
6, chromatographic condition
A. liquid phase part
Chromatographic column
Ether position: Hypersil BDS C18,4.6 * 250mm, 5 μ m
The ethyl acetate extract of 5% isoamylol: Aichrombond AQ C18,4.6 * 250mm, 5um
Moving phase
The ether position: acetonitrile-0.01% phosphate aqueous solution is the eluent gradient wash-out, and when retention time was 0min, 45min, 80min, 90min, 105min, the volume ratio of acetonitrile and 0.01% phosphate aqueous solution was respectively 8: 92,30: 70,50: 50,80: 20,80: 20;
The ethyl acetate extract of 5% isoamylol: acetonitrile-0.01% phosphate aqueous solution is the eluent gradient wash-out, when retention time is 0min, 50min, 80min, 100min, 110mi n, the volume ratio of acetonitrile and 0.01% phosphate aqueous solution was respectively 15: 85,30: 70,55: 45,100: 0,100: 0;
Flow velocity: 0.8ml/ minute
Detect wavelength: 280nm
Column temperature: 30 ℃
B. gas phase part
Chromatographic column Agilent HP-5,30m * 320m * 0.25 μ m
Injector temperature: 230 ℃
Fid detector temperature: 260 ℃
Heating schedule: initial 60 ℃, keep 12min, 6 ℃/min rises to 210 ℃, keeps 3min
Carrier gas is N2, flow velocity 1.0ml/min, current constant mode, split ratio 5: 1
Injector: G1888A head space automatic sampler, 85 ℃ of head space bottle temperature, 100 ℃ of sampling valve temperature, 115 ℃ of transmission line temperature, head space bottle equilibration time 15min, head space bottle pressurising time 0.5min, quantitatively ring filling time 0.2min quantitatively encircles equilibration time 0.5min, sample injection time 1.0min;
7, detect
A. liquid phase part
The ether position: precision is drawn ether position need testing solution and each 4 μ l injection liquid chromatograph of standard solution respectively, according to " 2000 editions appendix high effective liquid chromatography for measuring of Chinese pharmacopoeia get ether position finger-print and standard items finger-print
The ethyl acetate extract of 5% isoamylol: accurate respectively ethyl acetate extract test sample and each 4 μ l of standard solution that draw 5% isoamylol, inject liquid chromatograph, according to " 2000 editions appendix high effective liquid chromatography for measuring of Chinese pharmacopoeia, the ethyl acetate extract finger-print and the standard items finger-print of 5% isoamylol;
B. gas phase part
The Chinese strength wine of directly getting above-mentioned lot number seals in the headspace sampling bottle; Carry out assay determination by the head-space sampler auto injection, get gas phase part finger-print and standard items finger-print;
8, interpretation of result is judged
A. the gained lot number be detected in the 20041220 Chinese strength wine ether position test sample finger-prints with according to 10 identical characteristic peaks of finger-print; Wherein No. 7 peaks are forulic acid, and are consistent with the standard items finger-print; Test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient is 0.9899, greater than 0.9000, meets the correlated quality standard code that our company formulates.
B. the gained lot number is to have detected 9 characteristic peaks identical with reference fingerprint in the ethyl acetate extract test sample finger-print of 20041220 Chinese strength wine 5% isoamylol; Wherein No. 11 peaks are icariin, and are consistent with the standard items finger-print, and test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient should be 0.9617, greater than 0.9000; Meet the correlated quality standard code that our company formulates.
C. the gained lot number is to have detected 8 characteristic peaks identical with reference fingerprint in the 20041220 Chinese strength fume phase part test sample finger-prints; Wherein No. 7 peaks are acetic acid bornyl ester, and No. 8 peaks are eugenol, and are consistent with the standard items finger-print; Test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient is 0.9746, greater than 0.9000; Meet the correlated quality standard code that our company formulates.
1, sample source strength board company warehouse
2, the sample lot number 20050425
3, the preparation of standard solution is with embodiment 1
4, the preparation of need testing solution is with embodiment 1
5, key instrument and reagent are with embodiment 1
6, chromatographic condition is with embodiment 1
7, detection partly detects the liquid phase part and the gas phase of standard solution, this lot number need testing solution respectively by embodiment 1 same steps as and method, obtains ethyl acetate extract, the gas phase part finger-print of corresponding standard product, ether position, 5% isoamylol respectively;
8, interpretation of result is judged
A. the gained lot number is to have detected 10 characteristic peaks identical with reference fingerprint in the 20050425 Chinese strength wine ether position test sample finger-prints; Wherein No. 7 peaks are forulic acid, and are consistent with the standard items finger-print; Test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient is 0.9890, greater than 0.9000, meets the correlated quality standard code that our company formulates.
B. the gained lot number is to have detected 9 characteristic peaks identical with reference fingerprint in the ethyl acetate extract test sample finger-print of 20050425 Chinese strength wine 5% isoamylol; Wherein No. 11 peaks are the pubeseent epimedium herb glycosides, and are consistent with the standard items finger-print; Test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient is 0.9837, greater than 0.9000; Meet the correlated quality standard code that our company formulates.
C. the gained lot number is to have detected 8 characteristic peaks identical with reference fingerprint in the 20050425 Chinese strength fume phase part test sample finger-prints; Wherein No. 7 peaks are acetic acid bornyl ester, and No. 8 peaks are eugenol, and are consistent with the standard items finger-print; Test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient is 0.9953, greater than 0.9000; Meet the correlated quality standard code that our company formulates.
1, sample source strength board company warehouse
2, the sample lot number 20050723
3, the preparation of standard solution is with embodiment 1
4, the preparation of need testing solution is with embodiment 1
5, key instrument and reagent are with embodiment 1
6, chromatographic condition is with embodiment 1
7, detection partly detects the liquid phase part and the gas phase of standard solution, this lot number need testing solution respectively by embodiment 1 same steps as and method, obtains ethyl acetate extract, the gas phase part finger-print of corresponding standard product, ether position, 5% isoamylol respectively;
8, interpretation of result is judged
A. the gained lot number is to have detected 10 characteristic peaks identical with reference fingerprint in the 20050723 Chinese strength wine ether position test sample finger-prints; Wherein No. 7 peaks are forulic acid, and are consistent with the standard items finger-print; Test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient is 0.9972, greater than 0.9000, meets the correlated quality standard code that our company formulates.
B. the gained lot number is to have detected 9 characteristic peaks identical with reference fingerprint in the ethyl acetate extract test sample finger-print of 20050723 Chinese strength wine 5% isoamylol; Wherein No. 11 peaks are the pubeseent epimedium herb glycosides, and are consistent with the standard items finger-print; Test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient is 0.9427, greater than 0.9000; Meet the correlated quality standard code that our company formulates.
C. the gained lot number is to have detected 8 characteristic peaks identical with reference fingerprint in the 20050723 Chinese strength fume phase part test sample finger-prints; Wherein No. 7 peaks are acetic acid bornyl ester, and No. 8 peaks are eugenol, and are consistent with the standard items finger-print; Test sample finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient is 0.9930, greater than 0.9000; Meet the correlated quality standard code that our company formulates.
4 three batches of Chinese strength wine of embodiment (lot number 20041220,20050425,20050723) finger-print similarity evaluation
Each position finger-print of 20041220,20050425,20,050,723 3 lot numbers that embodiment 1, embodiment 2, embodiment 3 is measured is imported computer aided similarity evaluation system respectively and is calculated, its related coefficient is respectively the ether position: 0.9899 0.9890 0.9972, the ethyl acetate extract of 5% isoamylol: 0.9617 0.9837 0.942 7 gas phase parts: 0.9746 0.9953 0.9930, all greater than 0.9000, illustrate that mass discrepancy is little between three batches, constant product quality.
Claims (4)
1. detect Chinese strength wine method for quality with fingerprint pattern technology, may further comprise the steps:
The first step is set up Chinese strength wine reference fingerprint
(1) preparation of Chinese strength wine
Take off and state parts by weight of Chinese traditional medicine raw material Radix Astragali 10-15, saline cistanche 10-15, thizoma curculiginis 10-15, barrenwort 10-15, matrimony vine 10-15, Chinese yam 10-15, Radix Angelicae Sinensis 10-15, cloves 3-5, Chinese cassia tree 3-5,30-50 ° of distilled liquor diacolation with 50 times of total medicinal material amounts got percolate in 12-15 days, get percolate 70-75 part, it is an amount of to add 20 parts in 40% syrup, pure water 5-10 part, burnt sugar coloring respectively, allotment, filter, put under the normal temperature filtering and impurity removing, the smart filter of filter membrane ageing 3-6 month, can, promptly;
(2) preparation of Chinese strength wine liquid phase part reference substance solution
A. ether position
Get Chinese strength wine 100ml,, use water saturation extracted with diethyl ether 3 times with macroporous absorption chromatographic column desaccharification, each 20ml, combining extraction liquid, evaporate to dryness is settled to 2ml with tetrahydrofuran, membrane filtration, promptly;
B.5% the ethyl acetate extract of isoamylol
With ether position reference substance solution with the ethyl acetate extraction of water saturated 5% isoamylol 3 times, each 20ml, combining extraction liquid, evaporate to dryness is settled to 2ml with tetrahydrofuran, membrane filtration, promptly;
(3) preparation of Chinese strength fume phase part reference substance solution
Directly get Chinese strength wine 5ml in 10ml headspace sampling bottle, seal, promptly;
(4) preparation of standard solution
A. liquid phase part
It is an amount of to get the forulic acid standard items, makes the solution of 1mg/ml with 50% methyl alcohol; It is an amount of to get the icariin standard items, makes the solution of 1mg/ml with 50% methyl alcohol;
B. gas phase part
Dip in glass capillary and to get Bronyl acetate, eugenol standard items in right amount in the headspace sampling bottle, sealing, promptly;
(5) chromatographic condition
A. liquid phase part
Chromatographic column
The ether position: Hypersil C18 post,
The ethyl acetate extract of 5% isoamylol: Aichrombond AQ C18,
Moving phase
The ether position: acetonitrile-0.01% phosphate aqueous solution is the eluent gradient wash-out;
The ethyl acetate extract of 5% isoamylol: acetonitrile-0.01% phosphate aqueous solution is the eluent gradient wash-out;
Flow velocity: 0.8ml/ minute
Detect wavelength: 280nm
Column temperature: 30 ℃
B. gas phase part
Chromatographic column Agilent HP-5
Injector temperature: 230 ℃
Fid detector temperature: 260 ℃
Carrier gas is N2, flow velocity 1.0ml/min, split ratio 5: 1
Injector: G1888A head space automatic sampler;
(6) reference substance detects
A. liquid phase part
The ether position:
Accurate respectively Chinese strength wine liquid phase part ether position reference substance solution and each 4 μ l of standard solution of drawing inject liquid chromatograph, according to high effective liquid chromatography for measuring, promptly get Chinese strength wine ether position reference fingerprint and corresponding standard product finger-print;
The ethyl acetate extract of 5% isoamylol: accurate respectively ethyl acetate extract reference substance solution and each 4 μ l of standard solution that draw Chinese strength wine liquid phase part 5% isoamylol, inject liquid chromatograph, according to high effective liquid chromatography for measuring, promptly get the ethyl acetate extract reference fingerprint and the corresponding standard product finger-print of 5% isoamylol;
B. gas phase part
Respectively gas phase part reference substance solution and standard solution are carried out assay determination by the head-space sampler auto injection, promptly get gas phase part reference fingerprint and corresponding standard product finger-print;
In second step, set up Chinese strength wine finger-print common pattern collection of illustrative plates
Prepare 10 batches Chinese strength wine and standard items, need testing solution to be measured respectively by the preparation method of the preparation method of Chinese strength wine, Chinese strength wine liquid phase part and gas phase part reference substance solution, the preparation method of standard solution, and detect by same procedure one by one, obtain ethyl acetate extract finger-print, gas phase part finger-print and the corresponding standard product finger-print of the Chinese strength wine of each batch ether position finger-print, 5% isoamylol respectively; Import computer aided similarity evaluation system respectively and calculate, can draw the ethyl acetate extract of Chinese strength wine ether position, 5% isoamylol and the finger-print common pattern collection of illustrative plates of gas phase part;
In the 3rd step, test sample detects
1. the preparation of need testing solution
The ethyl acetate need testing solution and the gas phase part need testing solution that prepare liquid phase part ether position and 5% isoamylol with above-mentioned same procedure respectively;
2. prepare liquid phase part and gas phase part of standards product solution respectively with above-mentioned same procedure;
3. measure standard items and test sample to be measured with above-mentioned same procedure;
4, interpretation of result is judged
Should detect 10 characteristic peaks identical with reference fingerprint in the test sample finger-print of ether position, wherein No. 7 peaks are forulic acid, should be consistent with the standard items finger-print; Should detect 9 characteristic peaks identical in the ethyl acetate extract test sample finger-print of 5% isoamylol with reference fingerprint; Wherein No. 11 peaks are the pubeseent epimedium herb glycosides, should be consistent with the standard items finger-print; The finger-print of two parts and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient should be greater than 0.9000.
Should detect 8 characteristic peaks identical in the gas phase part test sample finger-print with reference fingerprint; Wherein No. 7 peaks are acetic acid bornyl ester, and No. 8 peaks are eugenol, should be consistent with the standard items finger-print; Its finger-print and corresponding common pattern the collection of illustrative plates auxiliary similarity evaluation system of machine as calculated calculate, and related coefficient should be greater than 0.9000.
2. according to claim 1ly detect Chinese strength wine method for quality with fingerprint pattern technology, it is characterized in that: the concrete grammar of the ethyl acetate extract reference substance solution preparation of described Chinese strength wine liquid phase part ether position and 5% isoamylol is, get Chinese strength wine 100ml, 40 ℃ of water-bath rotary evaporations are done near, be splined on 12mm * 300mm macroporous absorption chromatographic column that AB-8 is housed, water is eluted to the sugar-free reaction---and be that the Molish reaction is negative, back 80ml95% ethanol elution is collected eluent, evaporated under reduced pressure, add 20ml water, ultrasonicly make dissolving, and be transferred in the separating funnel, use water saturation extracted with diethyl ether 3 times earlier, each 20ml, combining extraction liquid, water bath method, be settled to 2ml with tetrahydrofuran, shake up, use 0.45 filtering with microporous membrane, promptly get ether position reference substance solution; The ethyl acetate extraction of 5% again that this solution with water is saturated isoamylol 3 times, each 20ml, combining extraction liquid, evaporated under reduced pressure is settled to 2ml with tetrahydrofuran, shakes up, and uses 0.45 filtering with microporous membrane, promptly gets the ethyl acetate extract reference substance solution of 5% isoamylol.
3. according to claim 1ly detect Chinese strength wine method for quality with fingerprint pattern technology, it is characterized in that: described liquid phase part ether position is the eluent gradient wash-out with acetonitrile-0.01% phosphate aqueous solution, its gradient is: when retention time is 0min, 45min, 80min, 90min, 105min, the volume ratio of acetonitrile and 0.01% phosphate aqueous solution was respectively 8: 92,30: 70,50: 50,80: 20,80: 20;
The ethyl acetate extract of 5% isoamylol: acetonitrile-0.01% phosphate aqueous solution is the eluent gradient wash-out, its gradient is: when retention time is 0min, 50min, 80min, 100min, 110min, the volume ratio of acetonitrile and 0.01% phosphate aqueous solution was respectively 15: 85,30: 70,55: 45,100: 0,100: 0.
4. according to claim 1ly detect Chinese strength wine method for quality with fingerprint pattern technology, it is characterized in that: described computer aided similarity evaluation system is meant the computer aided similarity evaluation system by the modernization of cmm center exploitation of Hunan Central South University that pharmacopoeia commission is recommended.
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