CN100376894C - Earthworm fingerprint spectrum establishment method and medicinal earthworm identification method - Google Patents
Earthworm fingerprint spectrum establishment method and medicinal earthworm identification method Download PDFInfo
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- 241000361919 Metaphire sieboldi Species 0.000 title claims abstract description 120
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000001228 spectrum Methods 0.000 title description 2
- 239000000463 material Substances 0.000 claims abstract description 55
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 33
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims abstract description 28
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000002904 solvent Substances 0.000 claims abstract description 5
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Abstract
The present invention relates to a method of establishing a Chinese traditional medicine earthworm HPLC fingerprint atlas and a standard fingerprint atlas. First, an earthworm medicinal material solution is prepared, wherein an amount of earthworm is taken, weighed precisely, dipped in 0.9% of physiological saline as a solvent at room temperature for 16 hours and centrifuged, a clear solution is taken to be filtered by a microporous filtering film (0.45 mum), and the obtained solution is used as the earthworm standard medicinal material solution. Second, a reference solution is prepared, wherein a proper quantity of hypoxanthine reference substances are taken, weighed precisely, dissolved and diluted by means of a mobile phase to be made into the reference solution. Finally, detection is carried out, wherein the earthworm standard medicinal material solution and the reference solution are respectively absorbed precisely and are filled in a liquid chromatograph to be detected according to a high performance liquid chromatography, and a chromatogram is recorded for 50 minutes. The high-performance liquid chromatography has the conditions that a chromatographic column uses octadecyl silane bonded silica gel as a bulking agent, the mobile phase is 0.01 mol/L of potassium dihydrogen phosphate solution and 50% of gradient eluting methanol solution, and a detecting wavelength is 254 nm. An earthworm sample fingerprint atlas to be detected is compared with a standard fingerprint atlas, and similarities and differences are compared. The method is simple, convenient and stable with high precision and good reproducibility, and is easily grasped.
Description
Technical field
The present invention relates to a kind of method for building up of earthworm finger-print and the discrimination method of earthworm medicinal material, specifically, is to adopt high performance liquid chromatography (HPLC) to set up the earthworm medicinal materials fingerprint and differentiate the earthworm medicinal material with finger-print.
Background technology
Earthworm is as traditional Chinese medicine, has heat-clearing arresting convulsion, vein relaxing, relievings asthma, the effect of diuresis, by Chinese Pharmacopoeia is recorded.The earthworm medicinal material kind that Chinese Pharmacopoeia records has four kinds, is that hard iron draws section's animal Pheretima aspergillum (Pheretima aspergillum (E.Perrier)) as medicinal principal item wherein, practises claiming LUMBRICUS.The main ingredient composition of earthworm is protein, amino acid, nucleic acid, enzyme, carbohydrate etc.Qualitative, the existing report of quantitative examination to composition such as protein, amino acid in the earthworm.Wherein the micromolecule composition is mainly hypoxanthine and nitrogen-containing compound.
Earthworm medicinal ingredient complexity, if single with wherein individually active components earthworm medicinal material inherent quality is described, have certain one-sidedness.Control the earthworm quality of medicinal material, preferably adopt its material group integral body to be controlled.That traditional Chinese medicine fingerprint is meant is common in certain Chinese crude drug or the Chinese patent drug, have distinctive certain class or the chromatogram of number constituents or the collection of illustrative plates of spectrum.Do not have under the clear and definite situation in the present stage Effective Components of Chinese Herb overwhelming majority, the quality for effective control Chinese crude drug or Chinese patent drug has great importance.The Japan main manufacturing enterprise of Chinese prescription medicine just adopts the high-efficiency liquid-phase fingerprint control of quality in enterprises in the eighties in 20th century.Germany, France find that the medical function of ginkgo biloba p.e is extract gained material group's mass action result in the process that ginkgo biloba p.e is developed jointly, and to the quality control of such integral body, also adopt the high-efficiency liquid-phase fingerprint method.In the plant herbal medicine guide of formulating U.S. FDA recent years clearly the method for quality control (FDA.Guidance of Industry:Botanical Drug (Draft) .2000August) of finger-print as the compounding substances group.Finger-print is become a consensus of the international community at present as Chinese herbal medicine and extraction of substance amount control method thereof.
Summary of the invention
So far, the method for building up about the earthworm finger-print does not but appear in the newspapers.For this reason, the inventor studies the method for building up of earthworm finger-print, and 10 batches of medicinal materials of earthworm are measured, and sets up the earthworm finger print measuring method.
The objective of the invention is by research earthworm water-soluble extractive HPLC finger-print, find out a kind of method of earthworm quality of medicinal material control, the method of the standard finger-print of earthworm is set up in establishment, whereby can be with the earthworm dactylogram as one of index of quality control and real and fake discrimination.The present invention also provides the standard finger-print of earthworm simultaneously.
The present invention implements through the following steps:
A kind of method for building up of earthworm finger-print comprises the steps:
(1) preparation of earthworm medicinal material solution: it is an amount of to get the earthworm medicinal material, and precision is weighed, and as solvent, preferably uses physiological saline with sodium chloride solution, extracts, and extract is through filtering with microporous membrane, as earthworm medicinal material solution;
(2) preparation of object of reference solution: it is an amount of to get the hypoxanthine reference substance, and precision is weighed, and with moving phase dissolving, dilution, makes object of reference solution;
(3) measure: accurate respectively earthworm medicinal material solution and the object of reference solution drawn, inject liquid chromatograph, according to high effective liquid chromatography for measuring, formulate the earthworm finger-print, wherein high-efficient liquid phase chromatogram condition is: the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and moving phase is phosphate-water-methanol gradient eluent, and the detection wavelength is 220nm～300nm, wherein, phosphate is preferably used potassium dihydrogen phosphate.
Wherein, described earthworm medicinal material, can be meal or earthworm section, for example can be all be 2000 ± 70um or 10 mesh sieves but to be mixed with to be that 355 ± 13um or 50 mesh sieves are no more than 20% meal by the sieve aperture internal diameter by the sieve aperture internal diameter, perhaps length is the earthworm section of 1cm, wherein preferably uses 1cm earthworm section.
Wherein, described extraction can be extracted or normal temperature (room temperature) immersion extraction for cold soaking, wherein preferably uses normal temperature (room temperature) to soak and extracts.Extraction time can be 12～36 hours, is preferably soaking at room temperature and extracts 16 hours.
Wherein, moving phase is phosphate-water-methanol different proportion solution, preferably presses the gradient proportioning as moving phase with potassium dihydrogen phosphate aqueous solution and methanol aqueous solution.
Wherein, detect wavelength and can select 230nm, 254nm, 294nm three medium wavelengths for use, wherein preferably use 254nm.
Wherein, set up the method for earthworm standard finger-print, comprise the steps:
(1) preparation of earthworm medicinal material solution: it is an amount of to get meal of earthworm or earthworm section, and precision is weighed, as solvent soaking at room temperature 16 hours, centrifugal with 0.9% physiological saline, gets supernatant and filters with miillpore filter (0.45um), as earthworm medicinal material solution;
(2) preparation of object of reference solution: it is an amount of to get the hypoxanthine reference substance, and precision is weighed, and with moving phase dissolving, dilution, makes object of reference solution;
(3) measure: accurate respectively earthworm medicinal material solution and the object of reference solution drawn, inject liquid chromatograph, according to high effective liquid chromatography for measuring, write down 50 minutes chromatograms, wherein high-efficient liquid phase chromatogram condition is: the chromatographic column octadecylsilane chemically bonded silica is a filling agent, moving phase is the gradient eluent of 0.01mol/L potassium dihydrogen phosphate (A) and 50% methyl alcohol (B), and the detection wavelength is 254 ± 1nm.
Wherein, described eluent gradient is: calculate A:0.01mol/L potassium dihydrogen phosphate, B:50% methyl alcohol by volume
| T(min) | A(％) | B(％) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 10 | 99 | 1 |
| 50 | 0 | 100 |
Wherein, column temperature is a room temperature; Flow velocity is 0.8～1ml/min, is preferably 0.8ml/min, and number of theoretical plate calculates by the hypoxanthine peak should be not less than 9000.
Wherein, above-mentioned described earthworm standard finger-print method for building up, in the earthworm standard finger-print that obtains, the common characteristic fingerprint peaks has 9, and its peak area summation accounts for more than 90% of total peak area.The peak area at non-total peak accounts for total peak area less than 10%.
Relative retention time and relative peak area with hypoxanthic chromatographic peak (label is S) are 1, and the common characteristic fingerprint peaks is in the resulting earthworm standard finger-print:
No. 3 peaks: relative retention time 0.528～0.557, relative peak area 0.089～0.657;
No. 4 peaks: relative retention time 0.596～0.639, relative peak area 0.023～0.126;
No. 5 peaks: relative retention time 0.779～0.799, relative peak area 0.017～0.188;
S peak (with reference to the peak): relative retention time 1, relative peak area 1;
No. 6 peaks: relative retention time 1.198～1.209, relative peak area 0.062～0.514;
No. 7 peaks: relative retention time 1.332～1.433, relative peak area 0.097～0.445;
No. 9 peaks: relative retention time 1.609～1.790, relative peak area 0.040～2.927;
No. 10 peaks: relative retention time 1.678～1.835, relative peak area 0.049～1.281;
No. 12 peaks: relative retention time 2.054～2.263, relative peak area 0.024～0.367.
Preferably, be 1 with the relative retention time and the relative peak area of hypoxanthic chromatographic peak (label is S), the common characteristic fingerprint peaks is in the resulting earthworm standard finger-print:
No. 3 peaks: relative retention time 0.543, relative peak area 0.284;
No. 4 peaks: relative retention time 0.618, relative peak area 0.070;
No. 5 peaks: relative retention time 0.792, relative peak area 0.067;
S peak (with reference to the peak): relative retention time 1, relative peak area 1;
No. 6 peaks: relative retention time 1.204, relative peak area 0.198;
No. 7 peaks: relative retention time 1.386, relative peak area 0.243;
No. 9 peaks: relative retention time 1.700, relative peak area 1.066～1.560;
No. 10 peaks: relative retention time 1.753, relative peak area 0.468;
No. 12 peaks: relative retention time 2.164, relative peak area 0.137.
Wherein, No. 3 the peak is highly stable, and peak shape is good; 4, No. 5 peaks are more stable, but peak value is less; No. 6 the peak is basicly stable, and peak shape is good, and peak value is little sometimes; The S peak is highly stable, and peak shape is fabulous; There is an acromion at No. 7 peaks when number of theoretical plate 9500 is following; No. 9 the peak is highly stable, and peak shape is fabulous; No. 10 peaks are a peak separately, the integral area joint account; No. 12 the peak is more stable, and peak value is little sometimes.
As another object of the present invention, the present invention also provides a kind of discrimination method of earthworm medicinal material, comprises the steps:
(1) finger-print of mensuration earthworm sample to be measured;
(2) with the finger-print and the contrast of earthworm standard finger-print of earthworm sample to be measured, screening meets the earthworm medicinal material of earthworm standard finger-print;
Wherein, step (1) is to measure according to above-mentioned described earthworm standard finger-print method for building up, and the earthworm standard finger-print of step (2) is above-mentioned described earthworm standard finger-print.
Advantage of the present invention mainly contains:
(1) the HPLC finger-print of setting up with earthworm water-soluble extractive, preferred earthworm physiological saline extract is being represented the most of pharmacologically active of earthworm, can characterize the quality of earthworm medicinal material effectively.The main active of earthworm is water soluble ingredients such as amino acid, polypeptide, polysaccharide, hypoxanthine, Lumbrokinase, so just have more representativeness with the water extract composition of earthworm as monitor control index.
(2) do as a wholely to treat with the whole fingerprint graph of earthworm, pay attention to each front and back that constitute fingerprint characteristic peak order and mutual relationship, pay attention to whole facial feature, both avoided judging the one-sidedness of earthworm total quality that having reduced again was the possibility of the artificial processing of requisite quality because of only measuring one, two chemical constitution.The present invention will contribute for quality and the curative effect that improves earthworm and prescribed preparation thereof for quality complete, that accurately estimate earthworm provides new reference standard.
(3) the present invention have that method is easy, stable, precision is high, favorable reproducibility, the characteristics that are easy to grasp.And the present invention only needs water normal temperature to extract, and sample preparation is greatly easy, has saved the cost and the time of check.
Description of drawings
Fig. 1: object of reference hypoxanthine HPLC collection of illustrative plates
Fig. 2: earthworm standard (contrast) finger-print (0～50 minute)
Embodiment
Below further specify the present invention by specific embodiment, but be not construed as limiting the invention.
Embodiment 1: the method for building up of earthworm standard finger-print
1, instrument and reagent
Successively test in the test at Hewlett-Packard's high performance liquid chromatograph and chromatographic work station HP1100, Tianjin, island high performance liquid chromatograph LC2010A.
Methyl alcohol is chromatographically pure, and water is distilled water, and potassium dihydrogen phosphate is pure for analyzing.
The hypoxanthine reference substance is the biochemical factory in Shanghai import packing product, and recording purity through the HPLC normalization method is 99.6%.
Earthworm medicinal material standard items are produced by the Guangxi Yulin, are accredited as hard iron through the institute for drug control, Heilongjiang Province and draw section's animal Pheretima aspergillum (Pheretima aspergillum (E.Perrier)), are numbered 11～20 respectively.
2, chromatographic condition
Chromatographic column: octadecylsilane chemically bonded silica;
Moving phase: the A:0.01mol/L potassium dihydrogen phosphate, B:50% methyl alcohol, gradient is:
| T(min) | A(％) | B(％) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 10 | 99 | 1 |
| 50 | 0 | 100 |
The detection wavelength is 254nm, and column temperature is a room temperature; Flow velocity is 0.8ml/min, and number of theoretical plate calculates by the hypoxanthine peak should be not less than 9000.
3, determining fingerprint pattern
(1) earthworm finger-print preparation
The preparation of object of reference solution: get an amount of 10mg of hypoxanthine reference substance, precision is weighed, and puts in the 100ml bottle, adds the 0.01mol/L potassium dihydrogen phosphate and makes dissolving, and be diluted to scale, shakes up, and makes object of reference solution;
The preparation of earthworm control medicinal material solution: get earthworm 1cm section 1g,, precision is weighed, and puts in the centrifuge tube, adds 0.9% physiological saline 10ml, and room temperature was placed 16 hours, and is centrifugal, gets supernatant and filters with miillpore filter (0.45um), as earthworm control medicinal material solution;
Accurate respectively object of reference solution 10ul and the earthworm standard solution 6ul of drawing injects liquid chromatograph, writes down 50 minutes chromatograms.Retention time and peak area with hypoxanthine chromatographic peak (S peak) are 1, calculate the relative retention time and the peak area ratio of each characteristic peak.
Object of reference hypoxanthine HPLC collection of illustrative plates and earthworm standard finger-print (0～50 minute) are seen Fig. 1 and 2 respectively.
(2) total peak determines
According to the correlation parameter that 10 batches of earthworm medicinal materials fingerprints provide, all occur in 50 minutes of the chromatographic peak of earthworm medicinal material.Relatively the chromatographic peak of each batch medicinal material has been determined 9 common characteristic fingerprint peakses, and its peak area summation accounts for more than 90% of total peak area.
Relative retention time and relative peak area with hypoxanthic chromatographic peak (label is S) are 1, and the retention time of common characteristic fingerprint peaks and peak area see Table 1 and 2 in the resulting earthworm standard finger-print.
Embodiment 2: the stability test of finger-print
According to finger-print 4stability determination operation, be test sample to be numbered 12 earthworm medicinal material, investigates 16 hours solution-stabilized, respectively relative retention time that has the peak and the peak area that relative peak area has regulation are added up, RSD is no more than 3%.Test sample normal temperature was stablized in 16 hours, met the technical requirement (seeing Table 3 and 4) of finger-print.
Embodiment 3: the precision test of finger-print
Operate according to finger-print precision prescriptive procedure, to be numbered 12 earthworm medicinal material is test sample, get 1 part, continuous sample introduction 5 times, respectively the relative retention time at total peak and the peak area that relative peak area has regulation are added up, RSD is no more than 3%, and the precision of whole detection system such as instrument is good, meets the technical requirement (seeing Table 5 and 6) of finger-print substantially.
Embodiment 4: the reappearance test of finger-print
Operate according to finger-print reappearance prescriptive procedure, to be numbered 12 earthworm medicinal material is test sample, get 5 parts, detect finger-print respectively, the relative retention time at total peak and the peak area that relative peak area has regulation are added up, RSD is no more than 3%, meets the technical requirement (seeing Table 7 and 8) of finger-print.
Embodiment 5: differentiate the earthworm medicinal material with the earthworm standard finger-print
The assay method of the finger-print of commercially available medicinal material (available from city's medicinal material company) with the method for building up of earthworm standard finger-print, writes down 50 minutes chromatograms.Relative retention time and relative peak area with hypoxanthic chromatographic peak (label is S) are 1, and be basic as differentiating with the earthworm standard finger-print of embodiment 1, and the difference of the finger-print of comparative sample and earthworm standard finger-print differentiates whether both are consistent.
Total peak retention time of table 1 earthworm medicinal material and relative retention time
The ratio (1) of total peak of table 2 earthworm medicinal material and non-total peak area
The ratio (2) of total peak of table 2 earthworm medicinal material and non-total peak area
*Non-total peak area number percent
Table 3 earthworm medicinal material stability test result (showing retention time and relative retention time)
Table 4 earthworm medicinal material stability test result (showing peak area and peak area ratio)
Table 5 earthworm medicinal material Precision test result (showing retention time and relative retention time)
Table 6 earthworm medicinal material Precision test result (showing peak area and peak area ratio)
Table 7 earthworm medicinal material reproducible test results (showing retention time and relative retention time)
Table 8 earthworm medicinal material reproducible test results (showing peak area and peak area ratio)
Each characteristic fingerprint peak relative retention time and peak area relative ratio among Fig. 2:
Relative retention time
No. 3 peaks: No. 0.5434 peak: No. 0.6185 peak: 0.792S peak: 1.000
No. 6 peaks: No. 1.2047 peaks: No. 1.3869 peaks: No. 1.70010 peaks: 1.753
No. 12 peaks: 2.164
Characteristic fingerprint peak-to-peak area relative ratio
No. 3 peaks: No. 0.2844 peak: No. 0.0705 peak: 0.067S peak: 1.000
No. 6 peaks: No. 0.1987 peak: No. 0.2439 peak: 1.333 ± No. 20%10 peaks: 0.468
No. 12 peaks: 0.137
Claims (7)
1. the method for building up of an earthworm finger-print comprises the steps:
(1) preparation of earthworm medicinal material solution: it is an amount of to get the earthworm medicinal material, and precision is weighed, and extracts as solvent with sodium chloride solution, and extract is through filtering with microporous membrane, as earthworm medicinal material solution;
(2) preparation of object of reference solution: it is an amount of to get the hypoxanthine reference substance, and precision is weighed, and with moving phase dissolving, dilution, makes object of reference solution;
(3) measure: accurate respectively earthworm medicinal material solution and the object of reference solution drawn, inject liquid chromatograph, according to high effective liquid chromatography for measuring, formulate the earthworm finger-print, wherein high-efficient liquid phase chromatogram condition is: the chromatographic column octadecylsilane chemically bonded silica is a filling agent, moving phase is phosphate-water-methanol gradient eluent, the detection wavelength is 220nm～300nm, eluent gradient is: calculate by volume, 100% 0.01mol/L potassium dihydrogen phosphate in the time of 0 minute, 100% 0.01mol/L potassium dihydrogen phosphate in the time of 5 minutes, 99% 0.01mol/L potassium dihydrogen phosphate and 1% 50% methyl alcohol in the time of 10 minutes, 100% 50% methyl alcohol in the time of 50 minutes.
2. method according to claim 1, wherein said sodium chloride solution are physiological saline.
3. the method for building up of an earthworm standard finger-print comprises the steps:
(1) preparation of earthworm medicinal material solution: it is an amount of to get meal of earthworm or earthworm section, and precision is weighed, as solvent soaking at room temperature 16 hours, centrifugal with 0.9% physiological saline, gets supernatant 0.45um filtering with microporous membrane, as earthworm medicinal material solution;
(2) preparation of object of reference solution: it is an amount of to get the hypoxanthine reference substance, and precision is weighed, and with moving phase dissolving, dilution, makes object of reference solution;
(3) measure: accurate respectively earthworm medicinal material solution and the object of reference solution drawn, inject liquid chromatograph, according to high effective liquid chromatography for measuring, write down 50 minutes chromatograms, wherein high-efficient liquid phase chromatogram condition is: the chromatographic column octadecylsilane chemically bonded silica is a filling agent, moving phase is the gradient eluent of 0.01mol/L potassium dihydrogen phosphate and 50% methyl alcohol, the detection wavelength is 254 ± 1nm, eluent gradient is: calculate by volume, 100% 0.01mol/L potassium dihydrogen phosphate in the time of 0 minute, 100% 0.01mol/L potassium dihydrogen phosphate in the time of 5 minutes, 99% 0.01mol/L potassium dihydrogen phosphate and 1% 50% methyl alcohol in the time of 10 minutes, 100% 50% methyl alcohol in the time of 50 minutes.
4. method according to claim 3, wherein, in the resulting earthworm standard finger-print, the common characteristic fingerprint peaks has 9, and its peak area summation accounts for more than 90% of total peak area.
5. according to claim 3 or 4 described methods, wherein, in the resulting earthworm standard finger-print, be 1 with the relative retention time and the relative peak area of hypoxanthic chromatographic peak, the common characteristic fingerprint peaks is selected from:
No. 3 peaks: relative retention time 0.528～0.557, relative peak area 0.089～0.657;
No. 4 peaks: relative retention time 0.596～0.639, relative peak area 0.023～0.126;
No. 5 peaks: relative retention time 0.779～0.799, relative peak area 0.017～0.188;
S number with reference to the peak: relative retention time 1, relative peak area 1;
No. 6 peaks: relative retention time 1.198～1.209, relative peak area 0.062～0.514;
No. 7 peaks: relative retention time 1.332～1.433, relative peak area 0.097～0.445;
No. 9 peaks: relative retention time 1.609～1.790, relative peak area 0.040～2.927;
No. 10 peaks: relative retention time 1.678～1.835, relative peak area 0.049～1.281;
No. 12 peaks: relative retention time 2.054～2.263, relative peak area 0.024～0.367.
6. method according to claim 5, wherein, the common characteristic fingerprint peaks is selected from:
No. 3 peaks: relative retention time 0.543, relative peak area 0.284;
No. 4 peaks: relative retention time 0.618, relative peak area 0.070;
No. 5 peaks: relative retention time 0.792, relative peak area 0.067;
S number with reference to the peak: relative retention time 1, relative peak area 1;
No. 6 peaks: relative retention time 1.204, relative peak area 0.198;
No. 7 peaks: relative retention time 1.386, relative peak area 0.243;
No. 9 peaks: relative retention time 1.700, relative peak area 1.066～1.560;
No. 10 peaks: relative retention time 1.753, relative peak area 0.468;
No. 12 peaks: relative retention time 2.164, relative peak area 0.137.
7. the discrimination method of an earthworm medicinal material comprises the steps:
(1) finger-print of mensuration earthworm sample to be measured;
(2) with the finger-print and the contrast of earthworm standard finger-print of earthworm sample to be measured, screening meets the earthworm medicinal material of earthworm standard finger-print;
Wherein, step (1) is to measure in accordance with the method for claim 3, and the earthworm standard finger-print of step (2) is the earthworm standard finger-print that the described method of claim 6 obtains.
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| CN101266233B (en) * | 2007-03-16 | 2011-02-23 | 株洲千金药业股份有限公司 | Zanthoxylum dissitum Hemsl medicinal materials discrimination method |
| CN107821336B (en) * | 2017-11-07 | 2021-05-11 | 广东省生物资源应用研究所 | Method for evaluating quality of individual and population of earthworm fry |
| CN109324141B (en) * | 2018-12-18 | 2022-05-03 | 广州白云山中药饮片有限公司 | Characteristic fingerprint spectrum of guangdong or processed products thereof, and construction method and application thereof |
| CN111272894A (en) * | 2020-02-28 | 2020-06-12 | 山西大学 | Construction method and application of earthworm 1H NMR fingerprint |
| CN114047268B (en) * | 2021-11-26 | 2023-07-07 | 华润三九医药股份有限公司 | Finger print of Guangdong earthworm medicinal preparation, construction method and content determination method thereof |
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| CN1403809A (en) * | 2001-09-04 | 2003-03-19 | 洪筱坤 | Establishment and application of Chinese medicine chaacteristic fingerprint spectrum |
| CN1437982A (en) * | 2003-01-29 | 2003-08-27 | 黑龙江省中医研究院 | Medicinal composition for treating mycoplasma pneumonia and preparation method and quality-control method |
| JP2004101477A (en) * | 2002-09-12 | 2004-04-02 | Yoshio Yamauchi | Two-dimensional high performance liquid chromatographic device and protein analyzer using the same |
| WO2005098414A1 (en) * | 2004-03-30 | 2005-10-20 | Euro-Celtique S.A | Methods for detecting 14-hydroxycodeinone and codeinone |
| WO2005116629A1 (en) * | 2004-05-26 | 2005-12-08 | Ajinomoto Co., Inc. | Method and apparatus for analyzing aminofunctional compound |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1403809A (en) * | 2001-09-04 | 2003-03-19 | 洪筱坤 | Establishment and application of Chinese medicine chaacteristic fingerprint spectrum |
| JP2004101477A (en) * | 2002-09-12 | 2004-04-02 | Yoshio Yamauchi | Two-dimensional high performance liquid chromatographic device and protein analyzer using the same |
| CN1437982A (en) * | 2003-01-29 | 2003-08-27 | 黑龙江省中医研究院 | Medicinal composition for treating mycoplasma pneumonia and preparation method and quality-control method |
| WO2005098414A1 (en) * | 2004-03-30 | 2005-10-20 | Euro-Celtique S.A | Methods for detecting 14-hydroxycodeinone and codeinone |
| WO2005116629A1 (en) * | 2004-05-26 | 2005-12-08 | Ajinomoto Co., Inc. | Method and apparatus for analyzing aminofunctional compound |
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