CN102654484B - Marsdenia tenacissima medicinal material, method for establishing fingerprints of marsdenia tenacissima medicinal material preparation and application of method - Google Patents
Marsdenia tenacissima medicinal material, method for establishing fingerprints of marsdenia tenacissima medicinal material preparation and application of method Download PDFInfo
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Abstract
The invention relates to a marsdenia tenacissima medicinal material, a method for establishing fingerprints of a marsdenia tenacissima medicinal material preparation,application of the method to quality control of the marsdenia tenacissima medicinal material and the preparation of marsdenia tenacissima medicinal material and discloses fingerprints of phenolic acids and steroid saponins in the medicinal material and the preparation of marsdenia tenacissima, and a measurement method and an application of the fingerprints. The quality of the medicinal material and the preparation of marsdenia tenacissima is controlled by using the fingerprints of the phenolic acids and the steroid saponins, therefore, the identity, the producing area and the quality of the marsdenia tenacissima are identified and controlled, so that the quality of the medicinal material and the preparation of the marsdenia tenacissima has an actually controllable standard, and stable quality of the medicinal material and the preparation of the marsdenia tenacissima is ensured. The method for controlling the quality of the marsdenia tenacissima through measuring the fingerprints of the phenolic acids and the steroid saponins in the medicinal material and the preparation of marsdenia tenacissima is simple, stable and reliable in operation.
Description
Technical field
The present invention relates to the method for quality control of a kind of Chinese medicinal material and preparation thereof, the particularly method for building up of CAULIS MARSDENIAE TENACISSIMAE medicinal material and preparation high performance liquid chromatography finger-print thereof, and the CAULIS MARSDENIAE TENACISSIMAE medicinal material that method obtains thus and the standard finger-print of preparation thereof.
Background technology
CAULIS MARSDENIAE TENACISSIMAE is the dry rattan of Asclepiadaceae (Asclep iadaceae) Marsdenia plant Rajmahal creeper [Marsdenia tenacissima (Roxb.) Wight et Arn], have another name called glaucescent fissistigma root, flat rattan, Marsdenia tenacissima, yellowish banks rose, large bitter rattan etc., begin to be loaded in < < the southern regions of the Yunnan Province book on Chinese herbal medicine > >, record in the drug standards > > of version < < Yunnan Province in 1974, version < < Chinese Pharmacopoeia > > in 1977 and version < < Chinese Pharmacopoeia > > in 2010.
CAULIS MARSDENIAE TENACISSIMAE bitter, cold nature.Enter lung, stomach, urinary bladder channel.Have clearing heat and detoxicatingly, relieving cough and asthma, eliminating stagnation to stop pain, relieves internal heat anticancer, dispels rheumatism, logical newborn diuresis, blood activating and promoting tissue regeneration, the function of lactagogue.CAULIS MARSDENIAE TENACISSIMAE root, rattan, stem, leaf are medicinal, cure mainly pneumonia, bronchitis, bronchial astehma, sphagitis, tonsillitis, cystitis, furuncle swelling toxin.Control sore, cough and breathe heavily, laryngalgia, stomachache.According to < < Chinese herbal medicine for preventing side, select > > to record: " to cure mainly tumour as cancer of the esophagus, cardia cancer, cancer of the stomach, leukaemia, Hodgkin's disease, cervical carcinoma, lung cancer etc.The double disease of controlling is as the infection of the upper respiratory tract, bronchitis, bronchial astehma, difficult urination ".
The method of controlling at present CAULIS MARSDENIAE TENACISSIMAE quality of medicinal material mainly comprises the methods such as Characters Identification, Microscopic Identification, thin layer evaluation, physics and chemistry evaluation and assay.Assay is mainly by utilizing contents of steroid saponin in spectrophotometry CAULIS MARSDENIAE TENACISSIMAE to reach the object of controlling CAULIS MARSDENIAE TENACISSIMAE quality of medicinal material.
Patent of invention CN100434088C discloses a kind of intravenous administration formulation extracting from CAULIS MARSDENIAE TENACISSIMAE, its preparation technology is made as medicinal extract by CAULIS MARSDENIAE TENACISSIMAE extract, in medicinal extract, add pharmaceutic adjuvant to become to be applicable to the formulation for drip-feed administration again, function cures mainly as clearing heat and detoxicating, resolving phlegm and softening hard masses, be mainly used in clinically cancer of the esophagus, cancer of the stomach, lung cancer, liver cancer, and can Combined with Radiotherapy, the auxiliary curing of chemotherapy.
The method of quality control Main Basis standard WS-10630 (ZD-0630)-2002 of current CAULIS MARSDENIAE TENACISSIMAE intravenous administration formulation, total solid in main detection parenteral solution, pH, protein, oxalates, resin, tanning, potassium ion, thermal source etc., and utilize total phenolic content the quality control index using it as parenteral solution in spectrophotometry parenteral solution.
Steroid saponin constituents is to have the generally acknowledged CAULIS MARSDENIAE TENACISSIMAE medicinal material of reported in literature and the main active of preparation.Yet CAULIS MARSDENIAE TENACISSIMAE medicinal material be take the index that contents of steroid saponin is quality control, its parenteral solution be take the index that total phenolic content is quality control, and certain single component content is measured global feature, the drug action that can not reflect medicinal material and parenteral solution completely, can not well control the quality of CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution thereof.
Summary of the invention
The present invention relates to the method for building up of CAULIS MARSDENIAE TENACISSIMAE medicinal material and preparation high performance liquid chromatography finger-print thereof, and the CAULIS MARSDENIAE TENACISSIMAE medicinal material that method obtains thus and the standard finger-print of preparation thereof.By measuring the finger-print of CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution thereof, effectively control the quality of CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution thereof, make up the deficiency of existing Quality Control Technology, thereby guaranteed validity, security, stability and the homogeneity of CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution thereof.
The technical solution adopted for the present invention to solve the technical problems is: by high performance liquid chromatography, set up the finger-print of CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution thereof, thereby effectively control the quality of CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution thereof.Specifically comprise the steps:
One, steroid saponin fingerprint and the method for quality control of CAULIS MARSDENIAE TENACISSIMAE medicinal material
1, the method for building up of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredients fingerprint
(a) preparation of test sample: get CAULIS MARSDENIAE TENACISSIMAE medicinal material appropriate, pulverize, sieve, precision takes medicinal powder 1g, and precision adds methyl alcohol 40ml, then weighs; 40KH
zultrasonic extraction is more than 10 minutes, naturally cooling, with methyl alcohol, supplies weight, and jolting mixes; Filter, get subsequent filtrate 20ml and volatilize, residue dissolves with methyl alcohol 1ml, through 0.45 μ m filtering with microporous membrane, obtains need testing solution.
(b) preparation of object of reference solution: precision takes Tenacissosides A(You Nanjing Shenghe Pharmaceutical Co., Ltd research institute's plant chamber and provides) 12mg, is placed in 50ml volumetric flask, adds methyl alcohol and dissolves and be diluted to scale, shakes up object of reference solution and get final product.
Tenacissosides A structural formula is:
(c) chromatographic condition: the chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent; Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～23min:75%A → 65%A, 25%B → 35%B; 23～48min:65%A → 50%A, 35%B → 50%B; 48～60min:50%A → 50%A, 50%B → 50%B; 60～90min:50%A → 20%A, 50%B → 80%B; 90～100min:20%A → 10%A, 80%B → 90%B; 100～101min:10%A → 75%A, 90%B → 25%B; 101～120min:75%A → 75%A, 25%B → 25%B; Column temperature: 35 ℃, flow velocity: 0.8ml/min.Detecting device: evaporative light-scattering detector; Atomization gas flow velocity: 1.5-3.5L/min; Drift tube temperature: 80-115 ℃.
(d) measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and records the chromatographic peak of 0～120 minute, obtains CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print.
(e) CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredient standard finger-print: according to method provided by the invention, determined CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredient standard finger-print, comprise 11 total peaks, wherein 4 (S) are Tenacissosides A contrast peak.These common characteristic peaks have formed the fingerprint characteristic of CAULIS MARSDENIAE TENACISSIMAE medicinal material jointly, standard finger-print for CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin constituents, 0.56 ± 10% (1), 0.59 ± 10% (2), 0.92 ± 10% (3), 1.00 ± 10% (4), 1.06 ± 10% (5), 1.09 ± 10% (6), 1.27 ± 10% the relative retention time of each characteristic peak is: and (7, S), 1.38 ± 10% (8), 1.41 ± 10% (9), 1.49 ± 10% (10), 1.96 ± 10% (11).
2. the method that CAULIS MARSDENIAE TENACISSIMAE quality of medicinal material is controlled
(1) mensuration of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print to be measured: get CAULIS MARSDENIAE TENACISSIMAE medicinal material to be measured, process and measure CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print to be measured by above-mentioned method;
(2) CAULIS MARSDENIAE TENACISSIMAE quality of medicinal material is controlled: utilize traditional Chinese medicine fingerprint area of computer aided similarity evaluation software, CAULIS MARSDENIAE TENACISSIMAE medicinal materials fingerprint and standard finger-print are compared, with 11 characteristic peaks of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin, calculate, it is qualified that similarity is not less than 0.85 quality of medicinal material.The present invention does as a whole treating by CAULIS MARSDENIAE TENACISSIMAE medicinal material, by comparing its characteristic peak, can find out the nuance between different medicinal materials, is suitable for discriminating and the control of the CAULIS MARSDENIAE TENACISSIMAE medicinal material true and false, the place of production, quality.
Two, the fingerprint of Tongguanteng Injection and method of quality control
1, the method for building up of Tongguanteng Injection liposoluble ingredient finger-print
(a) preparation of test sample: get Tongguanteng Injection, through 0.45 μ m membrane filtration, subsequent filtrate is need testing solution.
(b) preparation of object of reference solution: it is appropriate that precision takes chlorogenic acid reference substance, adds 50% methyl alcohol and makes every 1ml containing the solution of 20 μ g, obtains.
(c) chromatographic condition: the chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent; Mobile phase A is 0.05% phosphate aqueous solution, and Mobile phase B is 0.05% phosphoric acid acetonitrile solution, gradient elution: 0～20min:98%A → 96%A, 2%B → 4%B; 20～70min:96%A → 93%A, 4%B → 7%B; 70～110min:93%A → 90%A, 7%B → 10%B; 110～115min:90%A → 85%A, 10%B → 15%B; 115～116min:85%A → 98%A, 15%B → 2%B; Equilibration time 10 minutes; Flow velocity 1.0ml/min; Detecting device: UV-detector; Detect wavelength 300nm; 25 ℃ of column temperatures.Number of theoretical plate calculates and should be at least 6000 by chlorogenic acid peak.
(d) measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and records the chromatographic peak of 0～116 minute, obtains Tongguanteng Injection phenolic acid class finger-print.
(e) Tongguanteng Injection liposoluble ingredient standard finger-print: according to method provided by the invention, determined Tongguanteng Injection liposoluble ingredient standard finger-print, comprised 11 total peaks, wherein 5 (S) are chlorogenic acid contrast peak.These common characteristic peaks have formed the fingerprint characteristic of parenteral solution liposoluble ingredient jointly, standard finger-print for Tongguanteng Injection liposoluble ingredient, 0.26 ± 10% (1), 0.50 ± 10% (2), 0.55 ± 10% (3), 0.94 ± 10% (4), 1.00 ± 10% the relative retention time of each characteristic peak is: and (5, S), 1.10 ± 10% (6), 1.15 ± 10% (7), 1.25 ± 10% (8), 1.39 ± 10% (9), 1.45 ± 10% (10), 1.53 ± 10% (11).
2, the assay method of Tongguanteng Injection steroid saponin ingredients fingerprint
(a) preparation of test sample: get Tongguanteng Injection, through 0.45 μ m membrane filtration, subsequent filtrate is need testing solution.
(b) preparation of object of reference solution: precision takes Tenacigenoside A(You Nanjing Shenghe Pharmaceutical Co., Ltd research institute's plant chamber and provides) 12mg, is placed in 50ml volumetric flask, adds methanol constant volume, shakes up object of reference solution and get final product.
Tenacigenoside A structural formula is:
(c) chromatographic condition: the chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent; Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～110.0min:90%A → 44%A, 10%B → 56%B; 110.0～110.1min:44%A → 90%A, 56%B → 10%B; 110.1～120.0min:90%A → 90%A, 10%B → 10%B; Flow velocity 0.8ml/min; 35 ℃ of column temperatures.Detecting device: evaporative light-scattering detector; Atomization gas flow velocity: 1.5-3.5L/min; Drift tube temperature: 80-115 ℃.
(d) measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and records the chromatographic peak of 0～120 minute, obtains.
(e) Tongguanteng Injection steroid saponin ingredient standard finger-print: according to method provided by the invention, determined Tongguanteng Injection steroid saponin ingredient standard finger-print, comprise 8 total peaks, wherein 7 (S) are Tenacigenoside A contrast peak.These common characteristic peaks have formed the fingerprint characteristic of Tongguanteng Injection steroid saponin jointly, standard finger-print for parenteral solution steroid saponin constituents, 0.23 ± 10% (1), 0.36 ± 10% (2), 0.60 ± 10% (3), 0.62 ± 10% (4), 0.66 ± 10% (5), 0.97 ± 10% (6), 1.00 ± 10% the relative retention time of each characteristic peak is: and (7, S), 1.01 ± 10% (8).
3. the method for Tongguanteng Injection quality control
(1) mensuration of Tongguanteng Injection finger-print to be measured: get Tongguanteng Injection to be measured, process and measure Tongguanteng Injection phenolic acid class finger-print to be measured and/or steroid saponin finger-print by above-mentioned method;
(2) Tongguanteng Injection quality control: utilize traditional Chinese medicine fingerprint area of computer aided similarity evaluation software, Tongguanteng Injection finger-print and standard finger-print are compared, phenolic acid class finger-print calculates similarities with 11 characteristic peaks and is not less than 0.80, and steroid saponin calculates similarities with 8 characteristic peaks, and to be not less than 0.80 parenteral solution up-to-standard.The present invention does as a whole treating by parenteral solution, by comparing its characteristic peak, can find out the nuance between different batches, is suitable for the discriminating of Tongguanteng Injection quality and control.
Advantage of the present invention is:
1, the present invention adopts acetonitrile-water system, utilize the method for gradient elution to set up CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin, Tongguanteng Injection phenolic acid class, Tongguanteng Injection steroid saponin finger-print, the method is easy and simple to handle, reliable and stable, precision is high, and degree of separation is good.
2, CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution are quality control index mainly with chlorogenic acid.Yet certain single component content is measured the quality that can not reflect Chinese medicine completely.Fingerprint pattern technology is one of gordian technique of the modernization of Chinese medicine, it is the state-of-the-art technology that traditional Chinese medicine quality is controlled, we use the technology of finger-print effectively to control the quality of CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution thereof, make up the deficiency of existing Quality Control Technology, thereby guaranteed validity, security, stability and the homogeneity of CAULIS MARSDENIAE TENACISSIMAE medicinal material and parenteral solution thereof.
3, steroid saponin constituents is to have the generally acknowledged CAULIS MARSDENIAE TENACISSIMAE medicinal material of reported in literature and the main active of preparation thereof, the present invention, on the basis of phenolic acid class finger-print, further adopts the finger-print control CAULIS MARSDENIAE TENACISSIMAE medicinal material of steroid saponin constituents and the quality of parenteral solution thereof.
4, the present invention adopts the finger-print of phenolic acid class and steroid saponin two class compositions as the quality control method of Tongguanteng Injection, both avoided judging because only measuring one, two chemical composition the one-sidedness of preparation total quality, reduced again as the artificial possibility of processing of requisite quality, the present invention provides effective ways for quality complete, accurate evaluation Tongguanteng Injection, thereby the quality of product and curative effect are guaranteed.
5, to have aspect easy in the present invention, is easy to grasp, and stable, precision is high, the feature of favorable reproducibility.
Accompanying drawing explanation
Fig. 1 is CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredient standard finger-print
Fig. 2 is Tongguanteng Injection liposoluble ingredient standard finger-print
Fig. 3 is Tongguanteng Injection steroid saponin ingredient standard finger-print
Fig. 4 is CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print precision
Fig. 5 is CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print stability
Fig. 6 is CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print repeatability
Fig. 7 is Tongguanteng Injection phenolic acid class finger-print precision and stability
Fig. 8 is Tongguanteng Injection phenolic acid class finger-print repeatability
Fig. 9 is Tongguanteng Injection steroid saponin finger-print precision
Figure 10 is Tongguanteng Injection steroid saponin finger-print stability
Figure 11 is Tongguanteng Injection steroid saponin finger-print repeatability
Figure 12 is the CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredients fingerprint through different disposal
Figure 13 is CAULIS MARSDENIAE TENACISSIMAE medicinal material 14 batch sample steroid saponin ingredients fingerprints
Figure 14 is Tongguanteng Injection 11 batch sample liposoluble ingredient finger-prints
Figure 15 is Tongguanteng Injection 23 batch sample steroid saponin ingredients fingerprints
Embodiment:
Embodiment 1
The checking of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredients fingerprint methodology:
Chromatographic condition: chromatographic column: Agilent ZORBAX SB-C
18(250 * 4.6mm, 5 μ m); Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～23min:75%A → 65%A, 25%B → 35%B; 23～48min:65%A → 50%A, 35%B → 50%B; 48～60min:50%A → 50%A, 50%B → 50%B; 60～90min:50%A → 20%A, 50%B → 80%B; 90～100min:20%A → 10%A, 80%B → 90%B; 100～101min:10%A → 75%A, 90%B → 25%B; 101～120min:75%A → 75%A, 25%B → 25%B; Column temperature: 35 ℃, flow velocity: 0.8ml/min, detecting device: Alltech2000ES type evaporative light-scattering detector; Atomization gas flow velocity: 2.9L/min; Drift tube temperature: 106.5 ℃.Sample size 20 μ L.
Precision test
Get same need testing solution, continuous sample introduction 6 times, the relative retention time at calculated characteristics peak and relative peak area, RSD is less than respectively 0.04% and 1.4%, and similarity is greater than 0.95, shows that precision is good, in Table 1 and Fig. 4.
Table 1 CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin fingerprint similarity
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Contrast |
| S1 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S2 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S3 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S4 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S5 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S6 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| Contrast | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
Stability test
Get same need testing solution, respectively at the 0th, 2,4,8,16,24h measures, the relative retention time at calculated characteristics peak and relative peak area, RSD is less than respectively 0.05% and 2.86%, and similarity is greater than 0.95, shows that need testing solution is stable in 24h, in Table 2 and Fig. 5.
Table 2 CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin fingerprint similarity
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Contrast |
| S1 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S2 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S3 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S4 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 |
| S5 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 |
| S6 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 |
| Contrast | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
Replica test
Get with a collection of medicinal material with 6 parts of legal system available test solutions, measure the relative retention time at calculated characteristics peak and relative peak area, phase
Like degree, be greater than 0.95, RSD and be less than respectively 0.14% and 2.88%, show repeatability better, in Table 3 and Fig. 6.
Table 3 CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin fingerprint similarity
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Contrast |
| S1 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S2 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S3 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S4 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S5 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S6 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| Contrast | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
Embodiment 2
Tongguanteng Injection liposoluble ingredient fingerprint spectrum method is learned checking:
Chromatographic condition: chromatographic column: Agilent ZORBAX SB-C
18(250 * 4.6mm, 5 μ m); Mobile phase A is 0.05% phosphate aqueous solution, and Mobile phase B is 0.05% phosphoric acid acetonitrile solution, gradient elution: 0～20min:98%A → 96%A, 2%B → 4%B; 20～70min:96%A → 93%A, 4%B → 7%B; 70～110min:93%A → 90%A, 7%B → 10%B; 110～115min:90%A → 85%A, 10%B → 15%B; 115～116min:85%A → 98%A, 15%B → 2%B; Equilibration time 10 minutes; Flow velocity 1.0ml/min; Detect wavelength 300nm; 25 ℃ of column temperatures.
(lot number 200706301), continuous sample introduction 6 times (S1～S6), each 20 μ l, gained chromatogram integration calculates the similarity with reference fingerprint by finger-print software, all more than 0.95, in Table 4 and Fig. 7.
Table 4 Tongguanteng Injection phenolic acid class fingerprint similarity
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Reference fingerprint |
| S1 | 1.000 | 0.982 | 0.990 | 0.975 | 0.966 | 0.979 | 0.976 |
| S2 | 0.982 | 1.000 | 0.983 | 0.989 | 0.977 | 0.991 | 0.988 |
| S3 | 0.990 | 0.983 | 1.000 | 0.977 | 0.967 | 0.981 | 0.978 |
| S4 | 0.975 | 0.989 | 0.977 | 1.000 | 0.973 | 0.987 | 0.984 |
| S5 | 0.966 | 0.977 | 0.967 | 0.973 | 1.000 | 0.974 | 0.986 |
| S6 | 0.979 | 0.991 | 0.981 | 0.987 | 0.974 | 1.000 | 0.987 |
| Reference fingerprint | 0.976 | 0.988 | 0.978 | 0.984 | 0.986 | 0.987 | 1.000 |
(lot number 200706301) need testing solution 20 μ l, respectively at 0,2,4,6,8,10,12,16,18,24,30 hour mensuration (S1～S11) after preparation, gained chromatogram integration is the similarity with reference fingerprint by the calculating of finger-print software, all more than 0.95, show that sample kept stable in 30 hours, in Table 5 and Fig. 7.
Table 5 Tongguanteng Injection phenolic acid class fingerprint similarity
(lot number 200706301) 6 parts measured, and gained chromatogram integration calculates the similarity with reference fingerprint by finger-print software, all more than 0.95, in Table 6 and Fig. 8.
Table 6 Tongguanteng Injection phenolic acid class fingerprint similarity
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Reference fingerprint |
| S1 | 1.000 | 0.974 | 0.939 | 0.938 | 0.946 | 0.936 | 0.957 |
| S2 | 0.974 | 1.000 | 0.942 | 0.938 | 0.952 | 0.940 | 0.978 |
| S3 | 0.939 | 0.942 | 1.000 | 0.962 | 0.972 | 0.948 | 0.981 |
| S4 | 0.938 | 0.938 | 0.962 | 1.000 | 0.957 | 0.964 | 0.981 |
| S5 | 0.946 | 0.952 | 0.972 | 0.957 | 1.000 | 0.948 | 0.983 |
| S6 | 0.936 | 0.940 | 0.948 | 0.964 | 0.948 | 1.000 | 0.976 |
| Reference fingerprint | 0.975 | 0.978 | 0.981 | 0.981 | 0.983 | 0.976 | 1.000 |
Embodiment 3
The checking of Tongguanteng Injection steroid saponin ingredients fingerprint methodology:
Chromatographic condition: chromatographic column: Agilent ZORBAX SB-C
18(250 * 4.6mm, 5 μ m); Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～110.0min:90%A → 44%A, 10%B → 56%B; 110.0～110.1min:44%A → 90%A, 56%B → 10%B; 110.1～120.0min:90%A → 90%A, 10%B → 10%B; Flow velocity 0.8ml/min; 35 ℃ of column temperatures, detecting device: evaporative light-scattering detector; Atomization gas flow velocity: 2.9L/min; Drift tube temperature: 106.5 ℃, sample size 20 μ l.
Precision test
Get Tongguanteng Injection (lot number: 200810241), continuous sample introduction 6 times, press characteristic peak and calculate relative retention time and relative peak area, RSD is less than respectively 1% and 3%, gained chromatogram integration is the similarity with reference fingerprint by the calculating of finger-print software, all more than 0.95, in Table 7 and Fig. 9.
Table 7 Tongguanteng Injection steroid saponin fingerprint similarity
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Contrast |
| S1 | 1.000 | 0.999 | 0.998 | 0.998 | 0.995 | 0.997 | 0.999 |
| S2 | 0.999 | 1.000 | 0.998 | 0.999 | 0.999 | 0.996 | 0.999 |
| S3 | 0.998 | 0.998 | 1.000 | 0.999 | 0.999 | 0.996 | 0.998 |
| S4 | 0.998 | 0.999 | 0.999 | 1.000 | 0.999 | 0.999 | 0.999 |
| S5 | 0.995 | 0.999 | 0.999 | 0.999 | 1.000 | 0.996 | 0.998 |
| S6 | 0.997 | 0.996 | 0.996 | 0.999 | 0.996 | 1.000 | 0.998 |
| Contrast | 0.999 | 0.999 | 0.998 | 0.999 | 0.998 | 0.998 | 1.000 |
Stability experiment
Get Tongguanteng Injection (lot number: 200810241), respectively in 0h, 2h, 4h, 6h, 8h, 10h sample detection, press characteristic peak and calculate relative retention time and relative peak area, RSD is less than respectively 2% and 3%, gained chromatogram integration is the similarity with reference fingerprint by the calculating of finger-print software, all more than 0.95, in Table 8 and Figure 10.
Table 8 Tongguanteng Injection steroid saponin fingerprint similarity
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Contrast |
| S1 | 1.000 | 0.999 | 0.998 | 0.998 | 0.998 | 0.997 | 0.999 |
| S2 | 0.999 | 1.000 | 0.998 | 0.999 | 0.999 | 0.996 | 0.999 |
| S3 | 0.998 | 0.998 | 1.000 | 0.999 | 0.998 | 0.996 | 0.998 |
| S4 | 0.998 | 0.999 | 0.999 | 1.000 | 0.999 | 0.999 | 0.999 |
| S5 | 0.998 | 0.999 | 0.998 | 0.999 | 1.000 | 0.997 | 0.998 |
| S6 | 0.997 | 0.996 | 0.996 | 0.999 | 0.997 | 1.000 | 0.998 |
| Contrast | 0.999 | 0.999 | 0.998 | 0.999 | 0.998 | 0.998 | 1.000 |
Repeated experiment
Get Tongguanteng Injection (lot number: 200810241) 6 parts, press characteristic peak and calculate relative retention time and relative peak area, RSD is less than respectively 1% and 3%, and gained chromatogram integration is the similarity with reference fingerprint by the calculating of finger-print software, all more than 0.95, in Table 9 and Figure 11.
Table 9 Tongguanteng Injection steroid saponin fingerprint similarity
| ? | S1 | S2 | S3 | S4 | S5 | S6 | Contrast |
| S1 | 1.000 | 1.000 | 0.999 | 0.999 | 0.996 | 0.997 | 0.998 |
| S2 | 1.000 | 1.000 | 0.992 | 0.990 | 0.998 | 0.990 | 0.997 |
| S3 | 0.999 | 0.992 | 1.000 | 0.998 | 0.999 | 0.999 | 0.998 |
| S4 | 0.999 | 0.990 | 0.998 | 1.000 | 0.999 | 0.999 | 0.998 |
| S5 | 0.996 | 0.998 | 0.999 | 0.999 | 1.000 | 1.000 | 0.999 |
| S6 | 0.997 | 0.990 | 0.999 | 0.999 | 1.000 | 1.000 | 0.999 |
| Contrast | 0.998 | 0.997 | 0.998 | 0.998 | 0.999 | 0.999 | 1.000 |
Embodiment 4
The mensuration of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredients fingerprint
According to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2005), measure.
Chromatographic condition and system suitability: Agilent1200 series of high efficiency liquid chromatograph; Chromatographic column: Agilent ZORBAX SB-C
18(250 * 4.6mm, 5 μ m); Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～23min:75%A → 65%A, 25%B → 35%B; 23～48min:65%A → 50%A, 35%B → 50%B; 48～60min:50%A → 50%A, 50%B → 50%B; 60～90min:50%A → 20%A, 50%B → 80%B; 90～100min:20%A → 10%A, 80%B → 90%B; 100～101min:10%A → 75%A, 90%B → 25%B; 101～120min:75%A → 75%A, 25%B → 25%B; Column temperature: 35 ℃, flow velocity: 0.8ml/min.Detecting device: Alltech2000ES type evaporative light-scattering detector; Atomization gas flow velocity: 2.9L/min; Drift tube temperature: 106.5 ℃.Sample size 20 μ L.
The preparation of object of reference solution: precision takes Tenacissosides A(You Nanjing Shenghe Pharmaceutical Co., Ltd research institute's plant chamber and provides) 12mg, is placed in 50ml volumetric flask, adds methanol constant volume, shakes up object of reference solution and get final product.
The preparation of need testing solution: get CAULIS MARSDENIAE TENACISSIMAE medicinal material, pulverize, sieve, precision takes medicinal powder 1g, puts in 50ml conical flask, and precision adds methyl alcohol 40ml, then weighs; The ultrasonic extraction of 40KHZ 10 minutes, naturally cooling, with methyl alcohol, supply weight, jolting mixes; Filter, get subsequent filtrate 20ml and volatilize, residue dissolves with methyl alcohol 1ml.Through 0.45 μ m filtering with microporous membrane, obtain need testing solution.
Measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measure, record 0-120min chromatogram, obtain, by gained finger-print and standard finger-print comparison, through traditional Chinese medicine fingerprint similarity evaluation software, calculate, similarity is 0.974, sees T2 in Figure 12.
Embodiment 5
The mensuration of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredients fingerprint
According to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2005), measure.
Chromatographic condition and system suitability: Agilent1200 series of high efficiency liquid chromatograph; Chromatographic column: Agilent ZORBAX SB-C
18(250 * 4.6mm, 5 μ m); Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～23min:75%A → 65%A, 25%B → 35%B; 23～48min:65%A → 50%A, 35%B → 50%B; 48～60min:50%A → 50%A, 50%B → 50%B; 60～90min:50%A → 20%A, 50%B → 80%B; 90～100min:20%A → 10%A, 80%B → 90%B; 100～101min:10%A → 75%A, 90%B → 25%B; 101～120min:75%A → 75%A, 25%B → 25%B; Column temperature: 35 ℃, flow velocity: 0.8ml/min.Detecting device: Alltech2000ES type evaporative light-scattering detector; Atomization gas flow velocity: 1.5L/min; Drift tube temperature: 115.0 ℃.Sample size 20 μ L.
The preparation of object of reference solution: precision takes Tenacissosides A(You Shenghe medicine company research institute's plant chamber and provides) 12mg, is placed in 50ml volumetric flask, adds methanol constant volume, shakes up object of reference solution and get final product.
The preparation of need testing solution: get CAULIS MARSDENIAE TENACISSIMAE medicinal material, pulverize, sieve, precision takes medicinal powder 1g, puts in 50ml volumetric flask, and precision adds methyl alcohol 40ml, then weighs; The ultrasonic extraction of 40KHZ 90 minutes, naturally cooling, with methyl alcohol, supply weight, jolting mixes; Filter, get subsequent filtrate 20ml and volatilize, residue dissolves with methyl alcohol 1ml.Through 0.45 μ m filtering with microporous membrane, obtain need testing solution.
Measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measure, record 0-120min chromatogram, obtain, by gained finger-print and standard finger-print comparison, through traditional Chinese medicine fingerprint similarity evaluation software, calculate, similarity is 0.935, sees T3 in Figure 12.
Embodiment 6
The mensuration of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin ingredients fingerprint
According to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2005), measure.
Chromatographic condition and system suitability: Agilent1200 series of high efficiency liquid chromatograph; Chromatographic column: Agilent ZORBAX SB-C
18(250 * 4.6mm, 5 μ m); Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～23min:75%A → 65%A, 25%B → 35%B; 23～48min:65%A → 50%A, 35%B → 50%B; 48～60min:50%A → 50%A, 50%B → 50%B; 60～90min:50%A → 20%A, 50%B → 80%B; 90～100min:20%A → 10%A, 80%B → 90%B; 100～101min:10%A → 75%A, 90%B → 25%B; 101～120min:75%A → 75%A, 25%B → 25%B; Column temperature: 35 ℃, flow velocity: 0.8ml/min.Detecting device: Alltech2000ES type evaporative light-scattering detector; Atomization gas flow velocity: 3.5L/min; Drift tube temperature: 80.0 ℃.Sample size 20 μ L.
The preparation of object of reference solution: precision takes Tenacissosides A(You Shenghe medicine company research institute's plant chamber and provides) 12mg, is placed in 50ml volumetric flask, adds methanol constant volume, shakes up object of reference solution and get final product.
The preparation of need testing solution: get CAULIS MARSDENIAE TENACISSIMAE medicinal material, pulverize, sieve, precision takes medicinal powder 1g, puts in 50ml volumetric flask, and precision adds methyl alcohol 40ml, then weighs; The ultrasonic extraction of 40KHZ 60 minutes, naturally cooling, with methyl alcohol, supply weight, jolting mixes; Filter, get subsequent filtrate 20ml and volatilize, residue dissolves with methyl alcohol 1ml.Through 0.45 μ m filtering with microporous membrane, obtain need testing solution.
Measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measure, record 0-120min chromatogram, obtain, by gained finger-print and standard finger-print comparison, through traditional Chinese medicine fingerprint similarity evaluation software, calculate, similarity is 0.927, sees T4 in Figure 12.
Embodiment 7
The quality control of CAULIS MARSDENIAE TENACISSIMAE medicinal material
CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin determining fingerprint pattern to be measured:
According to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2005), measure.
Chromatographic condition and system suitability: Agilent1200 series of high efficiency liquid chromatograph; Chromatographic column: Agilent ZORBAX SB-C
18(250 * 4.6mm, 5 μ m); Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～23min:75%A → 65%A, 25%B → 35%B; 23～48min:65%A → 50%A, 35%B → 50%B; 48～60min:50%A → 50%A, 50%B → 50%B; 60～90min:50%A → 20%A, 50%B → 80%B; 90～100min:20%A → 10%A, 80%B → 90%B; 100～101min:10%A → 75%A, 90%B → 25%B; 101～120min:75%A → 75%A, 25%B → 25%B; Column temperature: 35 ℃, flow velocity: 0.8ml/min.Detecting device: Alltech2000ES type evaporative light-scattering detector; Atomization gas flow velocity: 2.9L/min; Drift tube temperature: 106.5 ℃.Sample size 20 μ L.
The preparation of object of reference solution: precision takes Tenacissosides A(You Shenghe medicine company research institute's plant chamber and provides) 12mg, is placed in 50ml volumetric flask, adds methanol constant volume, shakes up object of reference solution and get final product.
The preparation of need testing solution: get 14 batches of CAULIS MARSDENIAE TENACISSIMAE medicinal materials appropriate, pulverize, sieve, precision takes medicinal powder 1g, puts in 50ml round-bottomed flask, and precision adds methyl alcohol 40ml, then weighs; The ultrasonic extraction of 40KHZ 10 minutes, naturally cooling, with methyl alcohol, supply weight, jolting mixes; Filter, get subsequent filtrate 20ml, residue dissolves with methyl alcohol 1ml.Through 0.45 μ m filtering with microporous membrane, obtain need testing solution.
Measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and records 0-120min chromatogram, obtains, and sees Figure 13.
Similarity is calculated:
By gained finger-print and standard finger-print comparison, through traditional Chinese medicine fingerprint similarity evaluation software, to calculate, gained similarity result of calculation is in Table 10.
Table 10 CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin fingerprint similarity
| Sample number | S1 | S2 | S3 | S4 | S5 | S6 | S7 |
| Similarity | 0.983 | 0.977 | 0.975 | 0.985 | 0.899 | 0.862 | 0.700 |
| Sample number | S8 | S9 | S10 | S11 | S12 | S13 | S14 |
| Similarity | 0.920 | 0.961 | 0.959 | 0.985 | 0.985 | 0.913 | 0.944 |
CAULIS MARSDENIAE TENACISSIMAE evaluation of medical materials' quality:
CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print to be measured is all consistent with standard finger-print, occurs 11 characteristic peaks, and wherein 4 (S), for Tenacissosides A contrast peak, are shown in Figure 13.By gained finger-print and standard finger-print comparison, through similarity evaluation software, calculate, the similarity of sample 7 is lower than 0.85, and other is all more than 0.85, show that sample 7 does not meet quality requirements, and other CAULIS MARSDENIAE TENACISSIMAE medicinal material all conforms to quality requirements.
Embodiment 8
The quality control of Tongguanteng Injection
The mensuration of parenteral solution liposoluble ingredient finger-print
According to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2005), measure.
Chromatographic condition and system suitability: Agilent1200 series of high efficiency liquid chromatograph; The chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent, Agilent ZORBAX SB-C
18250 * 4.6mm5 μ m; Mobile phase A is 0.05% phosphoric acid solution, and Mobile phase B is 0.05% phosphoric acid acetonitrile solution, gradient elution: 0～20min:98%A → 96%A, 2%B → 4%B; 20～70min:96%A → 93%A, 4%B → 7%B; 70～110min:93%A → 90%A, 7%B → 10%B; 110～115min:90%A → 85%A, 10%B → 15%B; 115～116min:85%A → 98%A, 15%B → 2%B; Equilibration time 10 minutes; Flow velocity 1.0ml/min; Detect wavelength 300nm; 25 ℃ of column temperatures.Number of theoretical plate calculates and should be greater than 6000 by chlorogenic acid peak.
The preparation of object of reference solution: it is appropriate that precision takes chlorogenic acid reference substance, adds 50% methyl alcohol and makes every 1ml containing the solution of 20 μ g, obtains.
The preparation of need testing solution: get 11 batches of parenteral solutions, filter with miillpore filter (0.45 μ m), get subsequent filtrate, obtain.
Measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and records the chromatographic peak of 0 minute～116 minutes, obtains, and sees Figure 14.
Similarity is calculated:
By gained finger-print and standard finger-print comparison, through traditional Chinese medicine fingerprint similarity evaluation software, calculate, with standard finger-print comparison, gained similarity result of calculation is in Table 11.
Table 11 Tongguanteng Injection phenolic acid class fingerprint similarity
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 |
| Lot number | 200709121 | 200711301 | 200908051 | 200909071 | 200909141 | 200909161 |
| Similarity | 0.937 | 0.931 | 0.892 | 0.838 | 0.846 | 0.877 |
| Numbering | S7 | S8 | S9 | S10 | S11 | ? |
| Lot number | 200910161 | 200910281 | 201007051 | 201007071 | 201007201 | ? |
| Similarity | 0.903 | 0.881 | 0.889 | 0.935 | 0.914 | ? |
Tongguanteng Injection quality assessment:
Tongguanteng Injection phenolic acid class finger-print to be measured is all consistent with standard finger-print, occurs 11 characteristic peaks, and wherein 5 (S), for chlorogenic acid contrast peak, are shown in Figure 14.Through similarity evaluation software, calculate, similarity, all more than 0.8, shows that 11 batches of Tongguanteng Injections of survey all conform to quality requirements.
Embodiment 9
The quality control of Tongguanteng Injection
The mensuration of parenteral solution steroid saponin ingredients fingerprint
According to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2005), measure.
Chromatographic condition and system suitability: Agilent1200 series of high efficiency liquid chromatograph; The chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent (Agilent ZORBAX SB-C
18250 * 4.6mm, 5 μ m); Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～110.0min:90%A → 44%A, 10%B → 56%B; 110.0～110.1min:44%A → 90%A, 56%B → 10%B; 110.1～120.0min:90%A → 90%A, 10%B → 10%B; Flow velocity 0.8ml/min; Detect wavelength 300nm; 35 ℃ of column temperatures.Detecting device: Alltech2000ES evaporative light-scattering detector; Atomization gas flow velocity: 2.9L/min; Drift tube temperature: 106.5 ℃.Sample size 20 μ l.Theoretical cam curve: calculate and should be greater than 2000 by tenacigenoside A peak.
The preparation of need testing solution: get 23 batches of parenteral solutions, through 0.45 μ m membrane filtration, subsequent filtrate is need testing solution.
The preparation of object of reference solution: precision takes Tenacigenoside A(reference substance and provided by research institute of Nanjing Shenghe Pharmaceutical Co., Ltd plant chamber) 12mg, is placed in 50ml volumetric flask, adds methanol constant volume, shakes up reference substance solution and get final product.
Measure: accurate object of reference solution and each 20 μ l of need testing solution of drawing, injection liquid chromatography, measures and record chromatogram, sees Figure 15.
Similarity is calculated:
By gained finger-print and standard finger-print comparison, through traditional Chinese medicine fingerprint similarity evaluation software, to calculate, gained similarity result of calculation is in Table 12.
Table 12 Tongguanteng Injection steroid saponin fingerprint similarity
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | S7 |
| Lot number | 200808041 | 200808051 | 200808061 | 200808091 | 200808101 | 200808141 | 200809181 |
| Similarity | 0.990 | 0.993 | 0.992 | 0.992 | 0.985 | 0.989 | 0.98 |
| Numbering | S8 | S9 | S10 | S11 | S12 | S13 | S14 |
| Lot number | 200810241 | 200810271 | 200810281 | 200902181 | 200902201 | 200902231 | 200902241 |
| Similarity | 0.993 | 0.986 | 0.998 | 0.998 | 0.993 | 0.994 | 0.997 |
| Numbering | S15 | S16 | S17 | S18 | S19 | S20 | S21 |
| Lot number | 200903021 | 200903061 | 200903131 | 200903181 | 200903271 | 200903301 | 200904081 |
| Similarity | 0.990 | 0.992 | 0.931 | 0.996 | 0.995 | 0.994 | 0.985 |
| Numbering | S22 | S23 | ? | ? | ? | ? | ? |
| Lot number | 200904091 | 200904221 | ? | ? | ? | ? | ? |
| Similarity | 0.993 | 0.993 | ? | ? | ? | ? | ? |
Tongguanteng Injection quality assessment:
Tongguanteng Injection steroid saponin finger-print to be measured is all consistent with standard finger-print, occurs 8 characteristic peaks, and wherein 7 (S), for Tenacigenoside A contrast peak, are shown in Figure 15.Through similarity evaluation software, calculate, similarity, all more than 0.80, shows that 23 batches of Tongguanteng Injections of survey all conform to quality requirements.
Claims (2)
1. the method for building up of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print, is characterized in that: comprise the following steps:
(a) preparation of test sample: get CAULIS MARSDENIAE TENACISSIMAE medicinal material appropriate, pulverize, sieve, precision takes medicinal powder 1g, and precision adds methyl alcohol 40ml, then weighs; The ultrasonic extraction of 40KHz is more than 10 minutes, naturally cooling, with methyl alcohol, supplies weight, and jolting mixes; Filter, get subsequent filtrate 20ml and volatilize, residue dissolves with methyl alcohol 1ml, through 0.45 μ m filtering with microporous membrane, obtains need testing solution,
(b) preparation of object of reference solution: precision takes Tenacissosides A12mg, is placed in 50ml volumetric flask, adds methyl alcohol and dissolves and be diluted to scale, shake up, and object of reference solution and get final product,
(c) chromatographic condition: the chromatographic column that the octadecylsilane chemically bonded silica of take is filling agent; Mobile phase A is water, and Mobile phase B is acetonitrile, gradient elution: 0～23min:75%A → 65%A, 25%B → 35%B; 23～48min:65%A → 50%A, 35%B → 50%B; 48～60min:50%A → 50%A, 50%B → 50%B; 60～90min:50%A → 20%A, 50%B → 80%B; 90～100min:20%A → 10%A, 80%B → 90%B; 100～101min:10%A → 75%A, 90%B → 25%B; 101～120min:75%A → 75%A, 25%B → 25%B; Column temperature: 35 ℃, flow velocity: 0.8ml/min, detecting device: evaporative light-scattering detector; Atomization gas flow velocity: 1.5-3.5L/min; Drift tube temperature: 80-115 ℃,
(d) measure: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and records the chromatographic peak of 0～120 minute, obtains CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print,
Wherein, described CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print comprises 11 total peaks, wherein No. 4 peaks are Tenacissosides A contrast peak, and the relative retention time of each characteristic peak is: No. 1 peak: 0.56 ± 10%, No. 2 peaks: 0.59 ± 10%, No. 3 peaks: 0.92 ± 10%, No. 4 peaks: 1.00 ± 10%, No. 5 peaks: 1.06 ± 10%, No. 6 peaks: 1.09 ± 10%, No. 7 peaks: 1.27 ± 10%, No. 8 peaks: 1.38 ± 10%, No. 9 peaks: 1.41 ± 10%, No. 10 peaks: 1.49 ± 10%, No. 11 peaks: 1.96 ± 10%.
2. the method that CAULIS MARSDENIAE TENACISSIMAE quality of medicinal material is controlled, is characterized in that: comprise the following steps:
(a) mensuration of CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print to be measured: get CAULIS MARSDENIAE TENACISSIMAE medicinal material to be measured, by method claimed in claim 1, process and measure CAULIS MARSDENIAE TENACISSIMAE medicinal material steroid saponin finger-print to be measured,
(b) CAULIS MARSDENIAE TENACISSIMAE quality of medicinal material is controlled: by the steroid saponin finger-print of CAULIS MARSDENIAE TENACISSIMAE medicinal material to be measured and standard finger-print contrast, with characteristic peak calculating, it is qualified that similarity is not less than 0.85 quality of medicinal material,
Wherein said standard finger-print comprises 11 total peaks, wherein No. 4 peaks are Tenacissosides A contrast peak, and the relative retention time of each characteristic peak is: No. 1 peak: 0.56 ± 10%, No. 2 peaks: 0.59 ± 10%, No. 3 peaks: 0.92 ± 10%, No. 4 peaks: 1.00 ± 10%, No. 5 peaks: 1.06 ± 10%, No. 6 peaks: 1.09 ± 10%, No. 7 peaks: 1.27 ± 10%, No. 8 peaks: 1.38 ± 10%, No. 9 peaks: 1.41 ± 10%, No. 10 peaks: 1.49 ± 10%, No. 11 peaks: 1.96 ± 10%.
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| CN1709356A (en) * | 2005-06-02 | 2005-12-21 | 王衡新 | Anticancer Chinese medicine injection agent, and its preparing method and quality control method |
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Also Published As
| Publication number | Publication date |
|---|---|
| CN103792308B (en) | 2015-07-08 |
| CN102654484A (en) | 2012-09-05 |
| CN103792308A (en) | 2014-05-14 |
| CN104391065B (en) | 2016-03-30 |
| CN103884793A (en) | 2014-06-25 |
| CN104391065A (en) | 2015-03-04 |
| CN103884793B (en) | 2015-08-19 |
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