CN114689702A - Gel column HPLC fingerprint detection method for snake venom of Agkistrodon blossoms in Changbai mountain - Google Patents
Gel column HPLC fingerprint detection method for snake venom of Agkistrodon blossoms in Changbai mountain Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a fingerprint detection method for snake venom of a Changbai mountain Agkistrodon blomhoffii, which comprises the following steps: (1) preparation of test solution and control solution: respectively collecting venom test sample and venom control, dissolving in water, diluting, centrifuging, collecting supernatant, and filtering; (2) fingerprint detection: injecting the filtrate obtained in the step (1) into a liquid chromatograph, recording a chromatogram, and calculating the similarity by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, wherein gel for a chromatographic column is used as a filler; mobile phase: 50mM phosphate +300mM NaCl pH7.0, for isocratic elution. The method for detecting the snake venom fingerprint spectrum of the Changbai mountain Agkistrodon blomhoffii has strong operability, good reproducibility and high specificity, and can be used as a standard method for detecting and identifying the Changbai mountain Agkistrodon blomhoffii snake venom.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a fingerprint spectrum detection method of snake venom of a Changbai mountain Agkistrodon blomhoffii.
Background
The snake venom is prepared from the venom of Agkistrodon halys pallas (Agkistrodon, halis, pallas) parotid by squeezing and stimulating, freeze drying, and contains protein, polypeptide, enzymes, and other bioactive substances. The components of snake venom are very complex, and toxic components of different snake species, subspecies and even venom secreted by the same snake are different due to different seasons, producing areas, sexes, ages of the snakes and the like.
The fingerprint is derived from fingerprint identification science, is commonly used for traditional Chinese medicine research, and is a comprehensive, macroscopic and quantifiable identification means. It is established on the basis of systematic research on chemical components of Chinese medicinal materials, and is mainly used for evaluating the authenticity, the excellence and the stability of the quality of Chinese medicinal materials and semi-finished products of Chinese medicinal preparations, and the basic attributes are 'integrity' and 'fuzziness'. "integral" means the complete comparison of the chromatographic characteristics "face"; the emphasis of the ambiguity is the similarity of the reference substance and the fingerprint of the sample to be tested.
At present, the snake venom fingerprint detection method mainly comprises an RP-HPLC method, a gel chromatography method, a capillary electrophoresis method and the like. The HPLC has the characteristics of high separation efficiency, high selectivity, high detection sensitivity, high analysis speed, wide application range and the like. Therefore, the high performance liquid chromatography is the first choice of snake venom fingerprint spectrum technology.
Patent CN101539555A discloses a method for establishing a snake venom fingerprint, which adopts capillary electrophoresis to carry out separation and detection. However, this method is poor in reproducibility, low in sensitivity, and complicated in operation.
Juri Siigur et al ("Separation and analysis of peptides and proteins from Viper lebetin snake venom." Process Chemistry 2.1(2010): 109-. However, the method has poor peak shape and separation degree when used for detecting snake venom, and the smell of the mobile phase is large, so that the method is not beneficial to test operation.
Zuojun et al ("isolation and purification of thrombin from the venom of the northeast Baimei belly Snake venom", proceedings of Harbin institute 23.008(2002):59-61.) disclosed a method for analyzing purified fractions by HPLC using a TSK2000SW column with a mobile phase of 0.1mol/L phosphate buffer, pH6.8, and a detection wavelength of 280 nm. However, the method has the disadvantages of less peak number, poor separation degree and excessive mobile phase consumption when used for detecting snake venom, and is not favorable for saving cost.
Therefore, in order to better detect and identify the snake venom of the Changbai mountain Agkistrodon halys with white eyebrow and provide more comprehensive quality information of the snake venom of the Changbai mountain Agkistrodon with white eyebrow, a method for detecting the snake venom of the Changbai mountain Agkistrodon with white eyebrow with strong operability and good reproducibility is needed.
Disclosure of Invention
Aiming at the technical current situation, the invention provides a detection method of a snake venom fingerprint spectrum of a Changbai mountain Agkistrodon blomhoffii, which comprises the following steps:
(1) preparation of test solution and control solution: respectively dissolving and diluting venom test sample and venom reference sample with water, centrifuging, collecting supernatant, and filtering to obtain test solution and reference solution;
(2) fingerprint detection: respectively injecting the test solution and the reference solution obtained in the step (1) into a liquid chromatograph, recording a chromatogram, and calculating the similarity of the chromatograms of the test solution and the reference solution by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system;
the liquid chromatography conditions include:
and (3) chromatographic column: adopting gel as a filling agent;
mobile phase: 50mM phosphate +300mM NaCl pH 6.8-7.2, or 200mM phosphate pH6.8, or 200mM phosphate + 0.1% isopropanol pH 7.0; preferably 50mM phosphate +300mM NaCl pH 7.0;
isocratic elution was performed.
In the method of the present invention, as one embodiment, the step (1) further comprises: dissolving in water and diluting to obtain a solution containing 1-30 mg, preferably 2-20 mg, and most preferably 10mg per 1 ml.
In the method of the present invention, as one embodiment, the step (1) further comprises: the centrifugation condition is 3000-10000 rpm, preferably 3000rpm, 8-16 ℃, preferably 8 ℃, and the centrifugation is 15-20 min, preferably 15 min.
In the method of the present invention, as one embodiment, the step (2) further comprises: the chromatographic column is Waters Biosuite 2504 um UHR sec or TSKgel SuperSW 2000; preferably TSKgel SuperSW2000 with a specification of 4.6X 300mm, 4 um.
In the method of the present invention, as one embodiment, the step (2) further comprises: the column temperature is 23-27 ℃, and 25 ℃ is preferred; the flow rate is 0.33 to 0.37ml/min, preferably 0.35 ml/min.
In the method of the present invention, as one embodiment, the step (2) further comprises: the sample introduction volume is 5 μ l, the sample tray temperature is 8-25 deg.C, preferably 8-16 deg.C, most preferably 8 deg.C, and the detection wavelength is 280 nm.
In the method of the present invention, as one embodiment, the step (2) further comprises: the mobile phase 50mM phosphate +300mM NaCl pH7.0 is prepared by the following steps: taking 6.0g of sodium dihydrogen phosphate and 17.5g of sodium chloride, dissolving with water, and fixing the volume to 1L to obtain solution A; taking disodium hydrogen phosphate (Na)2HPO4·12H2O)17.9g and sodium chloride 17.5g, dissolving with water and fixing the volume to 1L to obtain solution B; and adjusting the pH value of the solution B to 7.0 by using the solution A.
In the method of the present invention, as one of embodiments, the method further comprises:
(1) preparation of test solution and control solution: dissolving and diluting venom test sample and venom reference substance of Agkistrodon halys pallas in Changbai mountain with water to obtain 10mg solution per 1ml, centrifuging at 3000rpm and 8 deg.C for 15min, collecting supernatant, and filtering to obtain test solution and reference substance solution;
(2) fingerprint detection: injecting 5 μ l of test sample solution and control solution into a liquid chromatograph, recording chromatogram, and calculating similarity between test sample spectrum and control sample spectrum by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system;
the chromatographic conditions include:
a chromatographic column: TSKgel SuperSW2000, 4.6X 300mm, 4 um;
flow rate: 0.35ml/min, sample injection volume, 5 mul;
column temperature: 25 ℃, sample pan temperature: 8 ℃;
detection wavelength: 280 nm;
mobile phase: 50mM phosphate +300mM NaCl pH 7.0; as one embodiment, the mobile phase is a mixed solution of 50mM phosphate and 300mM sodium chloride (6.0 g sodium dihydrogen phosphate and 17.5g sodium chloride are taken, dissolved in water and added to 1L to obtain solution A; and disodium hydrogen phosphate (Na) is taken2HPO4·12H2O)17.9g and sodium chloride 17.5g, dissolving with water and fixing the volume to 1L to obtain solution B; adjusting the pH value of the solution B to 7.0) by using the solution A;
and (3) an elution mode: isocratic elution, as an exemplary illustration, is performed for 22 minutes.
In the method of the invention, the fingerprint of the contrast of the Changbai mountain Agkistrodon blomhoffii venom comprises 8 characteristic peaks, and the specified value of relative retention time is as follows: 0.8037 (peak 1), 0.8926 (peak 2), 1.0570 (peak 4), 1.1438 (peak 5), 1.2415 (peak 6), 1.3822 (peak 7), 1.5017 (peak 8), the relative retention times of which should be within ± 5% of the stated values.
In the present invention, as one embodiment, the step (2) of detecting the fingerprint further includes: the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint spectrum is adopted for calculation, and the similarity between the sample spectrum and the reference substance spectrum is not less than 0.90.
As one embodiment, the step (2) further comprises that the similarity between the chromatogram of the test sample and the chromatogram of the control sample is not less than 0.95.
Compared with the existing detection method of the snake venom of the Agkistrodon blomhoffii from the Changbai mountain, the invention establishes a gel column HPLC fingerprint method, has strong operability, good reproducibility and high specificity, can provide more comprehensive quality information of the snake venom of the Agkistrodon blomhoffii from the Changbai mountain, and can be used as a standard method for detecting and identifying the snake venom of the Agkistrodon blomhoffii from the Changbai mountain.
Drawings
FIG. 1 is a different flow relative ratio chromatogram in example 2.
FIG. 2 is a graph showing the experimental spectrum of the precision experiment in example 3.
FIG. 3 is a graph showing the experimental pattern of the repetitive experiment in example 3.
FIG. 4 is the experimental profile-low temperature stability-of the stability experiment in example 3.
FIG. 5 is the experimental profile of the stability experiment in example 3-stability at room temperature.
FIG. 6 is a comparative overlay of the proprietary experiment in example 3.
Fig. 7 is a durability test pattern-column of example 3.
FIG. 8 is a graph of durability experiment of the method of example 3-flow rate, column temperature.
FIG. 9 is a graph of the durability experiment of the method of example 3-mobile phase pH.
FIG. 10 is an experimental map of the range survey in example 3.
Figure 11 is a comparison overlay of the feature maps of example 4.
FIG. 12 shows the consensus pattern of HPLC profiles of Agkistrodon halys venom of example 4 (gel column control).
Detailed Description
The following examples and test examples are intended to further illustrate the invention but are not intended to limit the effective scope of the invention in any way.
The instrument comprises the following steps:
reagent:
| serial number | Name of reagent | Specification of | Manufacturer of the |
| 1 | Sodium chloride | 500 g/bottle | Tianjin chemical |
| 2 | Anhydrous sodium dihydrogen phosphate | 500 g/bottle | TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd. |
| 3 | Disodium hydrogen phosphate dodecahydrate | 500 g/bottle | Tianjin |
| 4 | Purified water | —— | Self-made |
And (3) testing the sample:
comparison products: changbai mountain Agkistrodon halys venom, self-made.
Example 1 detection method of venom fingerprint of Agkistrodon blomhoffii on Changbai mountain
Sample (I)
And (3) testing the sample: changbai mountain white-eyebrow Agkistrodon halys snake venom (batch number: 20171207-4)
Comparison products: contrast medium of snake venom of Changbai mountain white-eyebrow Agkistrodon Halys (self-made)
(1) Preparation of test solution and control solution: dissolving and diluting venom test sample and venom reference substance of Agkistrodon halys pallas in Changbai mountain with water to obtain 10mg solution per 1ml, centrifuging at 3000rpm and 8 deg.C for 15min, collecting supernatant, and filtering to obtain test solution and reference substance solution;
(2) fingerprint detection: injecting 5 mul of test solution and reference solution into a liquid chromatograph, recording chromatogram, and calculating the similarity between the test sample spectrum and the reference sample spectrum by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, wherein the similarity between the test sample spectrum and the reference sample spectrum is not less than 0.90;
chromatographic conditions are as follows:
a chromatographic column: TSKgel SuperSW2000, 4.6X 300mm, 4 um; flow rate: 0.35ml/min, sample injection volume, 5 mul; column temperature: 25 ℃, sample pan temperature: 8 ℃, detection wavelength: 280 nm; mixing 50mM phosphate and 300mM sodium chloride (6.0 g sodium dihydrogen phosphate and 17.5g sodium chloride, dissolving in water to obtain solution A, and adding disodium hydrogen phosphate (Na)2HPO4·12H2O)17.9g and sodium chloride 17.5g, dissolving with water and fixing the volume to 1L to obtain solution B; adjusting the pH value of the solution B to 7.0 by using the solution A) as a mobile phase, and isocratically eluting for 22 minutes.
Example 2 screening of the Mobile phase
Sample (I)
And (3) testing the sample: changbai mountain white-eyebrow Agkistrodon halys snake venom (batch number: 20171207-4)
(1) Sample preparation: same as example 1 step (1)
(2) Fingerprint spectrum preparation: the mobile phase was adjusted as follows, otherwise the same as in (2) of example 1
Mobile phase 1:50mM phosphate +300mM NaCl pH7.0
Mobile phase 250 mM phosphate +300mM NaCl pH6.86
Mobile phase 3:200mM phosphate pH6.8
Mobile phase 4:200mM phosphate + 0.1% isopropanol pH7.0
Results and analysis
As can be seen from FIG. 1, the chromatographic behavior of mobile phases 1 and 2 is better than that of mobile phases 3 and 4, and the chromatographic parameters of mobile phases 1 and 2 are substantially the same, so that the optimal mobile phase system is selected to be 50mM phosphate +300mM NaCl pH7.0.
Example 3 methodological investigation of "detection method of venom fingerprint of Agkistrodon blomhoffii in Changbai mountain
1. Precision experiment
Sample (I)
And (3) testing the sample: changbai mountain white-eyebrow Agkistrodon halys snake venom (batch number: 20171207-4)
The experimental method comprises the following steps:
see example 1, the same sample is injected five times in parallel, measured legally, and examined for precision.
Results and analysis
The results are shown in figure 2, the peak (38k location) with RT of 10.242min is used as an internal reference peak, the relative retention time RSD of each main chromatographic peak is less than 0.1%, and the similarity of the measured chromatographic fingerprint and the obtained common pattern chart is respectively 1.000, 1.000 and 1.000 by calculation of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, which indicates that the instrument is stable and has good precision.
2. Repeatability test
Sample(s)
And (3) testing the sample: changbai mountain white-eyebrow Agkistrodon halys snake venom (batch number: 20171207-4)
The experimental method comprises the following steps:
referring to example 1, nine samples were prepared in parallel from the same batch of snake venom and tested by the method to examine the reproducibility of the method.
Results and analysis
TABLE 1 characteristic Peak relative Retention time (gel method)
TABLE 2 results of similarity (gel method)
The result is shown in tables 1-2 and figure 3, the No. 3 peak is taken as an internal reference peak, the relative retention time RSD of each main chromatographic peak is less than 0.1%, and the similarity of the measured chromatographic fingerprint and the obtained common mode diagram is more than 0.99 by using the calculation of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, so that the method has good repeatability.
3. Stability test
Sample(s)
And (3) testing the sample: changbai mountain white-eyebrow Agkistrodon halys snake venom (batch number: 20171207-4)
The experimental method comprises the following steps:
referring to the method of example 1, the same test solution was precisely aspirated, and the stability of the sample was examined within 96 hours of standing at low temperature (8 ℃) and within 24 hours of standing at room temperature after preparation, respectively.
Results and analysis of the experiments
The results are shown in figures 4-5, a peak (38k positioning) with RT of 10.242min is used as an internal reference peak, the relative retention time RSD of each main chromatographic peak under two investigation conditions is less than 0.5%, and the similarity of the measured chromatographic fingerprint and the obtained common pattern chart is more than 0.99 by calculation of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, so that the stability of the sample at 96 hours (8 ℃) and 24 hours (room temperature) after preparation is good.
4. Specificity experiments
Sample (I)
And (3) testing the sample: agkistrodon Halys venom of Changbai mountain Baimei (batch: 20171207-4), Agkistrodon saxatilis venom, Agkistrodon acutus venom, Jiangzhe Agkistrodon Halys venom, viper venom, Bothrops atrox venom, cobra venom, and bungarus fasciatus venom (batch: 20161227 from Naja-Xianglong-Yan-Xianling-Yangtong-Yangtang-snake Limited company; and batch No. 20170322 from other snake venom of Jiangxi Nongda special animal snake for demonstration base of snake culture)
Experimental methods
The method is the same as example 1, and different snake venoms are detected by the method, and the specificity of the method is examined.
TABLE 3 similarity results of Agkistrodon halys with other snakes
Results and analysis
The results are shown in Table 3 and FIG. 6. The result shows that the similarity of the finger print of other snake venoms and the snake venoms of the Agkistrodon blomhoffii is less than 0.9, and the specificity of the method is good.
5. Durability test
5.1 durability of different chromatography columns
Sample (I)
And (3) testing the sample: changbai mountain white-eyebrow Agkistrodon halys snake venom (batch number: 20171207-4)
The experimental method comprises the following steps:
and (4) respectively inspecting the durability of chromatographic column methods with the same specification and different brands.
Referring to the procedure of example 1, the column was adjusted to Waters Biosuite 2504 um UHR sec, TSKgel SuperSW2000 (4.6X 300mm, 4 um).
Results and analysis
The results are shown in fig. 7, the Waters gel column behaves substantially identically to the TSK column chromatography, and the process is robust.
5.2 durability of chromatographic conditions
Sample(s)
And (3) testing the sample: changbai mountain white-eyebrow Agkistrodon halys snake venom (batch number: 20171207-4)
The experimental method comprises the following steps:
referring to the method of example 1, the durability of the fingerprint spectrum detection method is examined when the flow rate, the column temperature and the mobile phase pH are slightly changed in the method. Except for the original conditions, the flow rate is checked to be 0.33ml/min and 0.37 ml/min; column temperature at 23 ℃ and 27 ℃; pH of mobile phase was examined at pH6.8, pH6.86, and pH 7.2.
Conclusion of the experiment
The result is shown in figures 8-9, the small change of the chromatographic condition has no influence on the whole outline of the fingerprint, and the durability is good.
6. Range of
Sample (I)
And (3) testing the sample: changbai mountain white-eyebrow Agkistrodon halys snake venom (batch number: 20171207-4)
Experimental methods
Referring to the method of example 1, similarity of snake venom fingerprints of test solution concentrations of 1, 2, 5, 10, 20 and 30mg/ml is respectively examined, and concentration ranges of the test solution are determined.
Results and analysis
Table 4 similarity results
The results are shown in figure 10 and table 4, the snake venom sample concentration is 1-30 mg/ml, the similarity of the snake venom fingerprint is more than 0.90, and the similarity is good.
Example 4 feature map and technical parameters
1. Establishment of snake venom characteristic map common mode of Agkistrodon halys (gel column method)
Sample(s)
And (3) testing the sample: the snake venom of Changbai mountain Agkistrodon halys with white eyebrow, 17 batches, the batch number is shown in the form of' test article
Experimental methods
Referring to example 1, the snake venom of Agkistrodon halys with different production areas and different seasons is measured according to the method, HPLC chart is recorded, data analysis processing is carried out by using the similarity evaluation system of traditional Chinese medicine fingerprint chromatogram of the national pharmacopoeia committee, a common mode is generated, and the similarity result of each test sample and the common mode is recorded.
Results and analysis
The results are shown in fig. 11-12, the similarity between the No. 1-17 snake venom samples and the common mode is respectively 0.986, 0.998, 0.999, 0.998 and 0.998, and is greater than 0.9, which indicates that the similarity of the snake venom characteristic maps is high, and indicates that the evaluation of the snake venom reference substance by the characteristic maps is reasonable.
2 calibration of characteristic peaks
Experimental methods
And the characteristic peak calibration is carried out on the basis of common peak calibration, fingerprint peaks of snake venom are locked in a 7-17 min common characteristic region for statistics, and 8 main common peaks are selected from 17 sample maps as characteristic peaks of a snake venom reference substance. And calculating the relative retention time of each characteristic peak by taking the No. 3 peak as an internal reference peak.
TABLE 5 relative Retention time Table of characteristic peaks
Results and analysis
The result of the analysis of the venom characteristic map of Agkistrodon halys gesii (Baimei Agkistrodon halys) shows that the relative retention time RSD of each characteristic peak in 17 batches of venom characteristic maps is less than 0.2%, and the relative retention time RSD accords with the relevant regulations (see Table 5, figures 11-12). The specified values are: 0.8037 (peak 1), 0.8926 (peak 2), 1.0570 (peak 4), 1.1438 (peak 5), 1.2415 (peak 6), 1.3822 (peak 7), 1.5017 (peak 8), the relative retention times of which should be within ± 5% of the stated values.
Conclusion of the experiment
The similarity evaluation is carried out by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, 8 characteristic peaks are selected to form the characteristic spectrum of the Agkistrodon halys venom, the result shows that the similarity of the characteristic spectrum is high, and a common mode is used as the identification standard of the Agkistrodon halys venom, so that more comprehensive quality control information can be provided. Experiments prove that the method has the characteristics of strong operability and good reproducibility, and can be used as an effective detection method for the quality of the Agkistrodon halys venom product.
Claims (11)
1. A fingerprint detection method for snake venom of Agkistrodon blomhoffii in Changbai mountain is characterized by comprising the following steps:
(1) preparation of test solution and control solution: respectively dissolving and diluting venom test sample and venom reference sample with water, centrifuging, collecting supernatant, and filtering to obtain test solution and reference solution;
(2) fingerprint detection: respectively injecting the test solution and the reference solution obtained in the step (1) into a liquid chromatograph, recording a chromatogram, and calculating the similarity of the chromatograms of the test solution and the reference solution by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system;
the liquid chromatography conditions include:
a chromatographic column: adopting gel as a filling agent;
mobile phase: 50mM phosphate +300mM NaCl pH 6.8-7.2, 200mM phosphate pH6.8 or 200mM phosphate + 0.1% isopropanol pH 7.0; preferably the mobile phase is 50mM phosphate +300mM NaCl pH 7.0;
isocratic elution was performed.
2. The method of claim 1, wherein step (1) further comprises: dissolving in water and diluting to obtain a solution containing 1-30 mg, preferably 2-20 mg, and most preferably 10mg per 1 ml.
3. The method of claim 1, wherein step (1) further comprises: the centrifugation condition is 3000-10000 rpm, preferably 3000rpm, 8-16 ℃, preferably 8 ℃, and the centrifugation is 15-20 min, preferably 15 min.
4. The method of claim 1, wherein step (2) further comprises: the chromatographic column is Waters Biosuite 2504 um UHR sec or TSKgel SuperSW 2000; preferably TSKgel SuperSW2000 with a specification of 4.6X 300mm, 4 um.
5. The method of claim 1, wherein step (2) further comprises: the column temperature is 23-27 ℃, and 25 ℃ is preferred; the flow rate is 0.33 to 0.37ml/min, preferably 0.35 ml/min.
6. The method of claim 1, wherein step (2) further comprises: the sample introduction volume is 5 μ l, the sample tray temperature is 8-25 deg.C, preferably 8-16 deg.C, most preferably 8 deg.C, and the detection wavelength is 280 nm.
7. The method of claim 1, wherein step (2) further comprises: the mobile phase 50mM phosphate +300mM NaCl pH7.0 is prepared by the following steps: taking 6.0g of sodium dihydrogen phosphate and 17.5g of sodium chloride, dissolving with water, and metering to 1L to obtain solution A; taking disodium hydrogen phosphate (Na)2HPO4·12H2O)17.9g and sodium chloride 17.5g, dissolving with water and fixing the volume to 1L to obtain solution B; and adjusting the pH value of the solution B to 7.0 by using the solution A.
8. The method of any one of claims 1 to 7, further comprising:
(1) preparation of test solution and control solution: dissolving venom test sample and control with water, diluting to obtain 10mg solution per 1ml, centrifuging at 3000rpm and 8 deg.C for 15min, collecting supernatant, and filtering to obtain test solution and control solution;
(2) fingerprint detection: injecting 5 μ l of test sample solution and control solution into a liquid chromatograph, recording chromatogram, and calculating similarity between test sample spectrum and control sample spectrum by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system;
the liquid chromatography conditions include:
and (3) chromatographic column: TSKgel SuperSW2000, 4.6X 300mm, 4 um;
flow rate: 0.35ml/min, sample injection volume, 5 mul;
column temperature: 25 ℃, sample pan temperature: 8 ℃;
detection wavelength: 280 nm;
mobile phase: 50mM phosphate +300mM NaCl pH 7.0;
isocratic elution.
9. The method of any one of claims 1 to 8, wherein the reference fingerprint in step (2) comprises 8 characteristic peaks, and the specified relative retention time is: 0.8037 (peak 1), 0.8926 (peak 2), 1.0570 (peak 4), 1.1438 (peak 5), 1.2415 (peak 6), 1.3822 (peak 7), 1.5017 (peak 8), the relative retention times of which should be within ± 5% of the stated values.
10. The method of claim 9, wherein the step (2) further comprises: the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint spectrum is adopted for calculation, and the similarity between the spectrum of the test sample and the spectrum of the reference substance is not less than 0.90.
11. The method according to claim 10, wherein the similarity between the test sample profile and the control profile in step (2) is not less than 0.95.
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| CN101539555A (en) * | 2008-03-18 | 2009-09-23 | 上海医药工业研究院 | Method for establishing snake poison fingerprint maps and fingerprint maps thereof |
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| CN101539555A (en) * | 2008-03-18 | 2009-09-23 | 上海医药工业研究院 | Method for establishing snake poison fingerprint maps and fingerprint maps thereof |
| CN101880656A (en) * | 2009-05-08 | 2010-11-10 | 北京赛生药业有限公司 | Agkistrodon halys venom thrombin and preparation method and application thereof |
| CN104498464A (en) * | 2014-12-24 | 2015-04-08 | 锦州奥鸿药业有限责任公司 | Method for extracting agkistrodon snake venom hemocoagulase from gloydius blomhoffi brevicaudus venom |
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