CN101726547A - Dendrobe chromatogram finger print measuring method - Google Patents
Dendrobe chromatogram finger print measuring method Download PDFInfo
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- CN101726547A CN101726547A CN200810218494A CN200810218494A CN101726547A CN 101726547 A CN101726547 A CN 101726547A CN 200810218494 A CN200810218494 A CN 200810218494A CN 200810218494 A CN200810218494 A CN 200810218494A CN 101726547 A CN101726547 A CN 101726547A
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Abstract
The invention discloses a dendrobe chromatogram finger print measuring method. The method comprises the following steps of: (1) cutting medical dendrobe, drying at low temperature, crushing, sieving for later use; (2) putting a proper amount of samples into a conical bottle, adding a proper amount of monohydric alcohol, performing ultrasonic treatment for 20 minutes, filtering, sprinkling filter slag by using the monohydric alcohol multiple times, combining filter liquors, and volatizing the filter liquors to near dryness; (3) dissolving by using the monohydric alcohol, fixing the volume, filtering by using a microporous filter membrane of 0.45 mum to be used as a sample solution; and (4) precisely sucking a comparison solution and the sample solution respectively, injecting into a liquid chromatograph for analyzing. The invention can obtain the chromatogram map conveniently and rapidly, and has easy obtaining of the raw materials, simple technology and advanced technique. According to the literature records, the variety of dendrobium plants used as the medical dendrobe is approximate to 100. The invention controls the product quality by using multiple components and multiple indexes, and has more reliable results, easy obtaining of the raw materials and simple technology compared with the quality control indexes.
Description
[technical field]
The present invention relates to a kind of application chromatographic fingerprinting technology, the chemical constitution of extracting in the medicinal dendrobium that contains phenols and flavonoids is worked out chromatogram finger print measuring method, so that the quality of each step in the purchasing of raw materials, product processes flow process is monitored.
[background technology]
The stem of noble dendrobium is an a class valuable Chinese crude drug commonly used, successive dynasties book on Chinese herbal medicine and each edition pharmacopeia are all recorded, Chinese and overseas scholars studies and explores the chemical constitution of the multiple stem of noble dendrobium, find that the contained chemical constitution type of this platymiscium is various, comprise compositions such as polysaccharide, alkaloid, flavonoids, quinones, sesquiterpenoids, Coumarins and steroidal glycosides compounds.In recent years, numerous scholars have carried out pharmacology activity research and clinical practice to separate the composition that obtains from multiple Dendrobium plant.Phenolic compound and flavone compound are one of main active in the stem of noble dendrobium, as isoflavonoid isoliquiritin in the stem of noble dendrobium, have the free radical of removing, oxidation resistant activity (Luo Dan etc., 2006), therefore, adopt modern quality control method, phenols and flavonoids chemical constitution quality control standard are that stem of noble dendrobium manufacturer emphasis is considered in the formulation stem of noble dendrobium.And the report that does not still adopt high performance liquid chromatogram (HPLC) fingerprint pattern technology that stem of noble dendrobium phenols and flavone compound are carried out quality control at present, therefore, this method is significant to the quality of control stem of noble dendrobium raw material and product.
[summary of the invention]
The objective of the invention is to overcome the weak point of existing Quality Control Technology, and method is simple, can convenient and swiftly obtain chromatogram, and can utilize the quality of this finger-print overall monitor stem of noble dendrobium, to guarantee the dendrobe chromatogram finger print measuring method of constant product quality.
The object of the present invention is achieved like this:
Dendrobe chromatogram finger print measuring method, step is as follows:
1., medicinal dendrobium cut off after, sieving for standby is pulverized in low temperature drying;
2., get an amount of sample in conical flask, add an amount of monohydroxy alcohol, ultrasonic 20min filters, filter residue with monohydroxy alcohol drip washing repeatedly, merging filtrate, filtrate is waved near and is done,
3., with monohydroxy alcohol dissolving, constant volume is used behind the 0.45 μ m filtering with microporous membrane as need testing solution again;
4., accurate reference substance solution and the need testing solution drawn respectively, inject liquid chromatograph, analyze;
Testing conditions is in the above-mentioned steps:
Chromatographic column: C
18Post (ODS post)
Flow velocity: 1ml/min
Detect wavelength: 254nm
Column temperature: 25 ℃
Moving phase: acetonitrile (A)-1 ‰ acetic acid (B).
(0 → 30min), (30 → 55min), 65 → 32%A (55 → 60min) for 32 → 65%A for gradient: 5% → 32%A.
Aforesaid dendrobe chromatogram finger print detection method, it is characterized in that sieving during step 1. was the 40-300 mesh sieve.
Aforesaid dendrobe chromatogram finger print measuring method, its feature described be in methyl alcohol, ethanol, isopropyl alcohol, n-propanol, the normal butyl alcohol any one in monohydroxy alcohol.
Aforesaid dendrobe chromatogram finger print measuring method is characterized in that described medicinal dendrobium is any one among HERBA DENDROBII Dendrobium nobile Lindl., dendrobium loddigesii Rolfe D.loddigesii Rolfe., HERBA DENDROBII D.chrysanthum Wall., stem of Eyeshaped Dendrobium D.fimbriatum Hook.Var.oculatumHook., the dendrobium candidum D.candidum Wall.ex Lindl..
The traditional method of quality control of the present invention and the stem of noble dendrobium is compared, and following outstanding advantage is arranged:
1, can conveniently obtain chromatogram, and starting material are easy to get, technology is simple, advanced technology.It is documented that the Dendrobium plant that can be used as medicinal dendrobium has nearly 100 kinds.Traditional method of quality control can not satisfy the requirement that above-mentioned sibling species is differentiated as methods such as appearance character evaluation, microscopical identification, thin layer discriminating, alkaloid and total polysaccharides assays.And the medical value of above-mentioned medicinal dendrobium and inequality has caused uneven, the shoddy situation of stem of noble dendrobium quality of medicinal material on the market.
The chromatographic fingerprints of Chinese materia medica technology is controlled the quality of product by multicomponent, many indexs, compares above-mentioned quality control index, and the result is more reliable, and starting material are easy to get, and technology is simple.
2, phenols and flavonoids are one of active component of medicinal dendrobium, so the present invention works out fingerprint pattern quality control method, can effectively characterize the quality of medicinal dendrobium.
3, the present invention adopts high performance liquid chromatography (HPLC) to work out the finger-print of medicinal dendrobium, compare with molecular biology method with employing vapor-phase chromatography (GC), method of operating of the present invention is simple, analysis speed is fast, cost is low, favorable reproducibility, is particularly suitable for the analysis of batch samples.
4, utilize assay method of the present invention, can obtain the finger-print chromatogram of stem of noble dendrobium sample.Relend and help computing machine similarity auxiliary evaluation software, sample drawing and total mode chart spectrum are compared, calculate, can estimate raw material variety, the place of production, true and false quality by similarity, and the stability of judging product quality.
[description of drawings]
Fig. 1 is Spherisorb ODS2C
18The finger-print of chromatographic column;
Fig. 2 .1 is PhenomenexC
18(250mm*4.6mm*5 μ m, Luna) finger-print of chromatographic column;
Fig. 2 .2 is Spherisorb ODS2C
18The finger-print of chromatographic column and Prodigy ODS3 chromatographic column;
Fig. 2 .3 is Prodigy ODS3 chromatographic column, Zorbax SBC
18Chromatographic column and PhenomenexC
18The finger-print of chromatographic column;
Fig. 3 is the blank assay chromatogram;
Fig. 4 is the chromatogram of need testing solution stability;
Fig. 5 is a need testing solution principal characteristic chromatogram;
Fig. 6 is a need testing solution precision chromatogram;
Fig. 7 is HERBA DENDROBII phenols, flavonoids finger-print;
Fig. 8 is the common pattern of 20 batches of HERBA DENDROBII phenols and flavones ingredient finger-print;
Fig. 9 is the finger-print of numbering 20 among Fig. 8;
Figure 10 is the finger-print of numbering 4 among Fig. 8;
Figure 11 is the finger-print of numbering 5 among Fig. 8;
Figure 12 is the spectrogram that obtains the 24.848min chromatographic peak behind the background deduction;
Figure 13 is the spectrogram that obtains the 25.281min chromatographic peak behind the background deduction.
[embodiment]
From object of experiment, we select and have compared different C in experiment
18(ODS post) chromatographic column: Spheris-orb ODS2 (150mm*4.6mm*5 μ m, waters) chromatographic column, Phenomenex C
18(250mm*4.6mm*5 μ m, Luna) chromatographic column, Zorbax SBC
18(250mm*4.6mm*5 μ m, Aglient) chromatographic column, Spherisorb ODS2 (250mm*4.6mm*5 μ m, waters) (250mm*4.6mm*5 μ m, Phenomenex) chromatographic column is to the influence of the separating effect of the chromatographic component of HERBA DENDROBII phenols and flavones ingredient for chromatographic column, Prodigy ODS3.Spherisorb ODS2 (150mm*4.6mm*5 μ m as shown in Figure 1, waters) chromatographic column, Spherisorb ODS2 post goes out peak number amount, peak height, peak type and better goes out all that the peak is intensive, degree of separation does not reach requirement of experiment far away, and therefore the chromatographic column of 150mm*4.6mm*5 μ m type is not finally adopted in experiment.The 250mm*4.6mm*5 μ m type chromatographic column good separating effect that adopts in the experimentation, appearance time is suitable, meets the chromatographic resolution optimization criteria, so adopt this type chromatographic column to experimentize.Compare (Fig. 2 .1,2.2,2.3) by different 250mm*4.6mm*5 μ m type chromatographic column gained chromatograms, final definite best Prodigy ODS3 (250mm*4.6mm*5 μ m of separating effect, Phenomenex) chromatographic column is carried out sample to be tested analysis (Spherisorb ODS2 (250mm*4.6mm*5 μ m, waters) the chromatographic column post is imitated and reduced serious hangover).Among Fig. 2 .3,1 is that (250mm*4.6mm*5 μ m, Phenomenex) chromatographic column 2 is Zorbax SBC to Prodigy ODS3
18(250mm*4.6mm*5 μ m, Aglient) chromatographic column; 3 is PhenomenexC
18(250mm*4.6mm*5 μ m, Luna) chromatographic column.
Desirable liquid chromatography mobile phase solvent should have low viscosity, good with the detecting device compatibility, be easy to obtain features such as pure product and hypotoxicity.Based on these several requirements, and, selected acetonitrile-1 ‰ acetate system as moving phase testing under the best precondition of gained HERBA DENDROBII phenols and flavones ingredient separating effect.Compare by experiment, this moving phase than simple acetonitrile-water system, the methanol-water system interference is little, composition peak number order is many, degree of separation is big, so select the experiment moving phase of acetonitrile-1 ‰ acetate system as the finger-print of setting up HERBA DENDROBII phenols and flavones ingredient for use.
HPLC has equal strength (isocratic) and gradient (gradient) wash-out dual mode.The equal strength wash-out is that the moving phase composition keeps constant in same analytical cycle, and it is less to be suitable for the component number, the sample that nature difference is little.Gradient elution is at the composition of an analytical cycle internal program control moving phase, as polarity of solvent, ionic strength and pH value etc., is used for analysis bank and divides the complex sample that number is many, nature difference is bigger.Adopt gradient elution to shorten analysis time, improve degree of separation, improve peak shape, improve detection sensitivity, but usually cause baseline wander and reduce reappearance.
Number is many, analysis time is long, considers the ageing and feasibility of repetitive operation in the practical application because HERBA DENDROBII phenols and flavones ingredient are analyzed, thereby tests the mode of first-selected gradient elution.Suitable analysis time when guaranteeing separating effect by the final income analysis result of allotment gradient elution program.For baseline wander and these situations of reduction reappearance that the gradient elution mode causes, methodology checking (seeing below) has been done in experiment, and the result is good.
For the sample that spectral absorption is arranged, the intensity of absorption signal changes with wavelength, only detects just under it has the wavelength of absorption maximum and may obtain maximum detection sensitivity.Select maximum absorption wavelength from full wavelength scanner 3D figure, it is many to take into account out the peak number amount, and the signal response value is big, the steady situation of baseline, and the 254nm wavelength is for detecting optimal wavelength.
Imitating because of post is the function of moving phase linear flow rate in the post, uses different flow velocitys can obtain different posts and imitates.For the fixed chromatographic column of a Gent, pursue optimum column efficiency, preferably use optimum flow rate.To internal diameter is the chromatographic column of 4.6mm, and flow velocity is generally selected 1ml/min.
Make blank assay with methyl alcohol, do not have component to flow out in the gained chromatographic fingerprinting, and baseline is very steady, as shown in Figure 3.
The chemical feature that the HERBA DENDROBII phenols that the method is set up and the chromatographic fingerprinting of flavones ingredient can be expressed this kind, i.e. uniqueness.
Stability experiment, the HERBA DENDROBII former state of getting a certain lot number is extracted by the method for drafting, and respectively at 0h, 1d and 2d different time are measured.On directly perceived, the chromatogram of these finger-prints is formed no significant change, as Fig. 4.Calculate the chromatographic fingerprinting that records at same instrument and the similarity of its common pattern collection of illustrative plates is respectively 0.9724,0.9877,0.9919 with similarity.
Repeated experiment is got the HERBA DENDROBII of same lot number, according to 4 parts of testing samples of method suggested preparation.Fig. 5 shows their chromatographic fingerprinting.Observe the chromatogram of these finger-prints and form no significant change.Calculate in same laboratory chromatographic fingerprinting that same instrument record and the similarity of its common pattern collection of illustrative plates is respectively 0.9881,0.9951,0.9953,0.9944,0.9952 with similarity based method (average).
Same need testing solution is got in the instrument precision experiment, continuous sample introduction 6 times, the consistance of investigation instrument precision and finger-print chromatographic component retention time.Fig. 6 is the chromatographic fingerprinting of precision test gained.The similarity coefficient (average) that calculates each finger-print and its common pattern collection of illustrative plates is 0.9876,0.9904,0.9951,0.9921,0.9860,0.9945.
According to above-mentioned detection, obtain final testing conditions:
Chromatographic condition:
Chromatographic column: Prodigy ODS3 (250mm*4.6mm*5 μ m, Phenomenex) chromatographic column
Flow velocity: 1ml/min
Detect wavelength: 254nm
Column temperature: 25 ℃
Moving phase: acetonitrile (A)-1 ‰ acetic acid (B).
Gradient: 5%-32%A (30min), 32-65%A (25min), 65-32%A (5min)
HERBA DENDROBII phenols and flavonoids need testing solution sample size are 20 μ l, and the sample component goes out the peak at 55min and finishes.
Instrument, according to situations such as the economic conditions of each manufacturer, instrument that each medicine inspection department had and the performance parameter of instrument, actual operability, recommend following instrument to be used for the finger-print research and the quality control of HERBA DENDROBII phenols and flavones ingredient: U.S. AgilentHP1100 high performance liquid chromatograph (G1322A type vacuum degassing machine, G1311A type quaternary pump, G1315A type diode array detector, G1328A type hand sampling device); B32005T type ultrasonic washing instrument.What select for use in the embodiment of the invention is the AgilentHP1100 high performance liquid chromatograph.
Experiment reagent: analyze pure methyl alcohol, trifluoroacetic acid aqueous solution, pure 36% acetate of analysis, analyze straight alcohol, redistilled water, isopropyl alcohol, n-propanol, normal butyl alcohol.
Data processing software, in whole HERBA DENDROBII phenols and flavonoids finger-print research process, be used for that the fingerprint spectrum data is handled and the instrument of quality assessment be medicine quality evaluated collection of illustrative plates/database (Extendable Data-base for QualityAssessment of Traditional Chinese Herbal Medicine) of writing of Central South University modernization of cmm center and computer aided similarity evaluation system (Computer Aided Similarity Evaluation, CASE).
The sample collection of HERBA DENDROBII phenols and flavones ingredient finger-print, 20 HERBA DENDROBII sample standard deviations are provided by the patentee, all pick up from the Chishui City, Guizhou Province.Wherein 10 samples are plucked HERBA DENDROBII GAP base, Yu Wanglong town; Other 10 samples are plucked in Changsha town head Xing Cun, third Anxiang San Focun, Aiwa village, third Anxiang, Ya Ling village, Wang Long town, Changsha town head Xing Cun, safflower village, Wang Long town, He Yun village, Changsha town, third Anxiang Bing Ancun, Changsha Zhen Gaodong village respectively, are revived the town.
(flow velocity 1ml/min detects wavelength: 254nm, column temperature: 25 ℃ to chromatographic condition Prodigy ODS3 for 250mm*4.6mm*5 μ m, Phenomenex) chromatographic column; Moving phase: acetonitrile (A)-1 ‰ acetic acid (B), gradient: 5%-32%A (0-30min), 32-65%A (30-55min), 65-32%A (55-60min)
The need testing solution sample size is 20 μ l, and the sample component goes out the peak at 55min and finishes.
The foundation of HERBA DENDROBII phenols and flavones ingredient finger-print is extracted 20 batches HERBA DENDROBII sample as need testing solution by working out extraction scheme.Obtain the finger-print of HERBA DENDROBII phenols and flavones ingredient with the HPLC-DAD method for combined use.After above-mentioned 20 batches of finger-print data being carried out baseline calibration and retention time drift calibration, as shown in Figure 7: 1 Changsha town; 2 third Anxiang San Focun; Aiwa village, 3 third Anxiang; Ya Ling village, 4 Wang Long town; 5 Changsha town head Xing Cun; Safflower village, 6 Wang Long town; He Yun village, 7 Changsha town; 8 third Anxiang Bing Ancun; Zhen Gaodong village, 9 Changsha; 10 revive the town; GAP base, 11-20 Wang Long town.
By the 20 batches of HERBA DENDROBII phenols and the related coefficient of flavones ingredient and the coefficient that is harmonious that calculates, as shown in table 1, wherein being numbered 18 samples of two samples and all the other of 4,5 compares and differs bigger, set up the meaning that common pattern then is contrary to common pattern foundation if choose the finger-print of 20 samples, so choose to remove and be numbered 4,5 18 outer samples are set up common pattern, as shown in Figure 8.
Table 1
| Numbering | Related coefficient (median) | Related coefficient (average) | Coefficient (median) is harmonious | Related coefficient (average) |
| ??1 | ??0.9339 | ??0.9424 | ??0.9364 | ??0.9432 |
| ??2 | ??0.9791 | ??0.9811 | ??0.9804 | ??0.9823 |
| ??3 | ??0.9310 | ??0.9389 | ??0.9354 | ??0.9431 |
| ??4 | ??0.8997 | ??0.9035 | ??0.9059 | ??0.9097 |
| ??5 | ??0.8407 | ??0.8540 | ??0.8514 | ??0.8659 |
| ??6 | ??0.9443 | ??0.9479 | ??0.9478 | ??0.9517 |
| ??7 | ??0.9303 | ??0.9386 | ??0.9341 | ??0.9434 |
| ??8 | ??0.9383 | ??0.9495 | ??0.9421 | ??0.9534 |
| ??9 | ??0.9460 | ??0.9452 | ??0.9491 | ??0.9495 |
| ??10 | ??0.9467 | ??0.9521 | ??0.9501 | ??0.9557 |
| ??11 | ??0.9719 | ??0.9691 | ??0.9735 | ??0.9706 |
| ??12 | ??0.9804 | ??0.9774 | ??0.9814 | ??0.9791 |
| Numbering | Related coefficient (median) | Related coefficient (average) | Coefficient (median) is harmonious | Related coefficient (average) |
| ??13 | ??0.9767 | ??0.9742 | ??0.9781 | ??0.9760 |
| ??14 | ??0.9830 | ??0.9804 | ??0.9840 | ??0.9818 |
| ??15 | ??0.9667 | ??0.9663 | ??0.9687 | ??0.9682 |
| ??16 | ??0.9824 | ??0.9825 | ??0.9833 | ??0.9838 |
| ??17 | ??0.9862 | ??0.9856 | ??0.9870 | ??0.9865 |
| ??18 | ??0.9695 | ??0.9694 | ??0.9712 | ??0.9717 |
| ??19 | ??0.9802 | ??0.9817 | ??0.9814 | ??0.9829 |
| ??20 | ??0.9821 | ??0.9821 | ??0.9830 | ??0.9835 |
As shown in table 2 by the related coefficient that calculates 20 batches of HERBA DENDROBII phenols and flavones ingredient and common pattern and the coefficient that is harmonious.Observe finger-print (as Fig. 8, Figure 10, shown in Figure 11), the sample collection of illustrative plates that is numbered 4 (as shown in figure 10), 5 (as shown in figure 11) has pattern to differ bigger together, this species diversity clearly, so cause the related coefficient of itself and common pattern and the coefficient that is harmonious less than normal; On the contrary, observe finger-print (as shown in Figure 9), being numbered 20 sample collection of illustrative plates has pattern extremely similar together, and it all reaches more than 0.98 with the coefficient that is harmonious with the related coefficient of common pattern.
Table 2
| Numbering | Related coefficient (median) | Related coefficient (average) | Coefficient (median) is harmonious | Related coefficient (average) |
| ??1 | ??0.9245 | ??0.9328 | ??0.9273 | ??0.9343 |
| ??2 | ??0.9830 | ??0.9830 | ??0.9842 | ??0.9841 |
| ??3 | ??0.9320 | ??0.9379 | ??0.9365 | ??0.9421 |
| ??4 | ??0.8930 | ??0.8998 | ??0.8997 | ??0.9063 |
| ??5 | ??0.8307 | ??0.8375 | ??0.8428 | ??0.8505 |
| ??6 | ??0.9453 | ??0.9490 | ??0.9490 | ??0.9527 |
| ??7 | ??0.9422 | ??0.9429 | ??0.9455 | ??0.9471 |
| ??8 | ??0.9483 | ??0.9509 | ??0.9516 | ??0.9546 |
| Numbering | Related coefficient (median) | Related coefficient (average) | Coefficient (median) is harmonious | Related coefficient (average) |
| ??9 | ??0.9416 | ??0.9432 | ??0.9454 | ??0.9476 |
| ??10 | ??0.9570 | ??0.9577 | ??0.9598 | ??0.9609 |
| ??11 | ??0.9624 | ??0.9658 | ??0.9646 | ??0.9675 |
| ??12 | ??0.9745 | ??0.9766 | ??0.9761 | ??0.9783 |
| ??13 | ??0.9828 | ??0.9802 | ??0.9839 | ??0.9816 |
| ??14 | ??0.9766 | ??0.9783 | ??0.9781 | ??0.9798 |
| ??15 | ??0.9583 | ??0.9623 | ??0.9609 | ??0.9645 |
| ??16 | ??0.9871 | ??0.9870 | ??0.9878 | ??0.9879 |
| ??17 | ??0.9844 | ??0.9856 | ??0.9855 | ??0.9865 |
| ??18 | ??0.9714 | ??0.9723 | ??0.9732 | ??0.9744 |
| ??19 | ??0.9851 | ??0.9854 | ??0.9860 | ??0.9864 |
| ??20 | ??0.9830 | ??0.9836 | ??0.9841 | ??0.9848 |
The spectrogram that obtains the 24.848min chromatographic peak behind the background deduction is a solvent with methyl alcohol as shown in figure 12, and the ultraviolet spectrum of flavones generally has two main absorption bands between 240-400nm, and the absorption band that is in the 290-380nm district claims to be with I, and the 240-280nm district is band II.Figure 12 and Figure 13 show, the absorption band of sample is all arranged at 275nm and 330nm place, according to aforementioned can simple and easy qualitative 24.848min and the 25.281min chromatographic peak be Flavonoid substances.
By above-mentioned experiment, draw stem of noble dendrobium finger print measuring method of the present invention, step is as follows:
1., medicinal dendrobium cut off 2-3cm after, 60 ℃ of dryings are pulverized, and cross 80 mesh sieves, and are standby;
2., get the 2g sample in conical flask, add 20ml methyl alcohol and soak, ultrasonic 20min, Buchner funnel filters, filter residue is with methyl alcohol drip washing 3 times, each 3ml, merging filtrate, filtrate is waved near and is done;
3., use dissolve with methanol, constant volume 2ml is again with dissolving as need testing solution behind the 0.45 μ m filtering with microporous membrane;
4., precision is drawn reference substance solution and need testing solution respectively, injects liquid chromatograph, promptly;
Testing conditions is in the above-mentioned steps:
Chromatographic column: Phenomenex Prodigy ODS3 (250mm*4.6mm*5 μ m) chromatographic column
Flow velocity: 1ml/min
Detect wavelength: 254nm
Column temperature: 25 ℃
Moving phase: acetonitrile (A)-1 ‰ acetic acid (B).
Gradient: 5%-32%A (0-30min), 32-65%A (30-55min), 65-32%A (55-60min).
The present invention can also be with the finger-print and the auxiliary similarity evaluation software comparison of machine as calculated of reference substance common pattern collection of illustrative plates of need testing solution.
Experiment reagent: analyze pure methyl alcohol (Hanbon Sci. ﹠ Tech. Co., Ltd.), trifluoroacetic acid aqueous solution (Hanbon Sci. ﹠ Tech. Co., Ltd.), analyze pure 36% acetate (chemical reagent factory of Hunan Normal University), analyze straight alcohol (Hanbon Sci. ﹠ Tech. Co., Ltd.), redistilled water (HPLC is pure); 0.45 μ m PTFE filers, isopropyl alcohol, n-propanol, normal butyl alcohol.
Adopt the 50ml conical flask in the present embodiment.
Medicinal dendrobium can be a kind of arbitrarily among HERBA DENDROBII Dendrobium nobile Lindl., dendrobium loddigesii Rolfe D.loddigesii Rolfe., HERBA DENDROBII D.chrysanthum Wall., stem of Eyeshaped Dendrobium D.fimbriatumHook.Var.oculatum Hook., the dendrobium candidum D.candidum Wall.ex Lindl..The assay method step of above-mentioned medicinal dendrobium, process conditions, testing conditions, data processing software are all identical.
Claims (4)
1. dendrobe chromatogram finger print measuring method, step is as follows:
1., medicinal dendrobium cut off after, sieving for standby is pulverized in low temperature drying;
2., get an amount of sample in conical flask, add an amount of monohydroxy alcohol, ultrasonic 20min filters, filter residue with monohydroxy alcohol drip washing repeatedly, merging filtrate, filtrate is waved near and is done,
3., with monohydroxy alcohol dissolving, constant volume is used behind the 0.45 μ m filtering with microporous membrane as need testing solution again;
4., accurate reference substance solution and the need testing solution drawn respectively, inject liquid chromatograph, analyze.
Testing conditions is in the above-mentioned steps:
Chromatographic column: C
18Post (ODS post)
Flow velocity: 1ml/min
Detect wavelength: 254nm
Column temperature: 25 ℃
Moving phase: acetonitrile (A)-1 ‰ acetic acid (B).
(0 → 30min), (30 → 55min), 65 → 32%A (55 → 60min) for 32 → 65%A for gradient: 5% → 32%A.
2. dendrobe chromatogram finger print detection method according to claim 1, it is characterized in that sieving during step 1. was the 40-300 mesh sieve.
3. dendrobe chromatogram finger print measuring method according to claim 1, its feature described be in methyl alcohol, ethanol, isopropyl alcohol, n-propanol, the normal butyl alcohol any one in monohydroxy alcohol.
4. dendrobe chromatogram finger print measuring method according to claim 1 is characterized in that described medicinal dendrobium is any one among HERBA DENDROBII Dendrobium nobile Lindl., dendrobium loddigesii Rolfe D.loddigesii Rolfe., HERBA DENDROBII D.chrysanthum Wall., stem of Eyeshaped Dendrobium D.fimbriatum Hook.Var.oculatum Hook., the dendrobium candidum D.candidum Wall.ex Lindl..
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| CN103604877A (en) * | 2013-10-29 | 2014-02-26 | 安徽农业大学 | Construction method and applications of high performance liquid chromatography (HPLC) fingerprint of alkaloid compounds of dendrobe |
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| CN105259269A (en) * | 2015-11-05 | 2016-01-20 | 北京蓝标一成科技有限公司 | Establishing method for dendrobe characteristic fingerprint spectrum |
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