CN113866309B - HPLC fingerprint spectrum establishment method and application of 11 bufogenin components in venenum bufonis - Google Patents
HPLC fingerprint spectrum establishment method and application of 11 bufogenin components in venenum bufonis Download PDFInfo
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Abstract
The invention belongs to the technical field of analysis and detection, and particularly relates to an HPLC (high performance liquid chromatography) fingerprint spectrum establishment method and application of 11 bufogenin components in toad venom. The invention further applies the fingerprint spectrum method to the quality detection of the multi-batch toad venom, and applies the similarity analysis, the principal component analysis and the cluster analysis to more comprehensively and effectively evaluate the internal quality of the toad venom, thereby providing basis and reference for the basic research and the clinical application of the toad venom.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to an HPLC fingerprint spectrum establishment method and application of 11 bufogenin components in toad venom.
Background
In China, toads have had a medicinal history for thousands of years, and ancient books on toads can be traced back to the Tang Dynasty's treatise on herb Property. Bufo Bufo gargarizans Cantor and Bufo melanostictus Schneider are two most important Bufo gargarizans in China, and comprise various medicinal parts such as venenum bufonis, cutis Bufonis, toad cortex and the like. At present, the toad venom becomes a II-grade wild medicinal material which is important to protect in China, and 88 adult preparations (329 batches) in 2020 edition of ministerial standards and pharmacopoeia of the people's republic of China contain the toad venom.
Bufonis venenum (Bufonis venenum) is dried product of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider of Bufo siccus, and has effects of removing toxic substance, relieving pain, inducing resuscitation and refreshing mind. Modern pharmacological studies show that active ingredients in the toad venom, such as bufalin, cinobufagin, resibufogenin and the like, have obvious curative effects on the aspects of tumor resistance, heart strengthening, pain relief, inflammation resistance, immunoregulation and the like. The pharmacopoeia of the people's republic of China (2020 edition) uses three bufogenin components, bufogenin, cinobufagin and resibufogenin, as index components for quality control of Bufonis venenum. The side chain at C17 position of the compound is connected with a six-membered unsaturated lactone ring (alpha-pyran ring), which is a key group of the compound for exerting physiological activity. However, according to the literature report, the existing research simultaneously measures 8 bufogenin components in the toad venom at most by using an HPLC method. However, as a traditional rare Chinese medicine in China, the toad venom has been separated and identified into hundreds of chemical components, and no report on simultaneous quantification of multiple components of more than 8 components exists at present.
Because the traditional Chinese medicine components are complex, the quality detection through a single component is time-consuming and labor-consuming, and the internal quality of the traditional Chinese medicine is difficult to be fully reflected. As a comprehensive and quantifiable identification means, the traditional Chinese medicine fingerprint spectrum can comprehensively reflect the types and the quantities of chemical components contained in the traditional Chinese medicine, thereby realizing the overall description and evaluation of the quality of the traditional Chinese medicine and becoming one of the effective means for controlling the quality of the traditional Chinese medicine at present. From 2006, the traditional Chinese medicine fingerprint is used for ingredient research of a venenum bufonis medicinal material, and a research method is established from the aspects of primordial origin, producing areas, preparations and the like to evaluate the quality of the venenum bufonis. However, these studies lack systematic evaluation by multi-index, multi-component, multi-analytical means, and the established fingerprint contains a small amount of active ingredients.
Disclosure of Invention
Aiming at the problems, the invention provides an HPLC fingerprint establishing method and application of 11 bufogenin components in toad venom. The invention establishes HPLC fingerprint spectrums of 11 bufogenin components, and comprehensively evaluates the quality of 12 batches of toad venom by combining content determination, similarity analysis, main component analysis and cluster analysis.
The invention relates to a method for establishing HPLC fingerprint spectra of 11 bufogenin components in toad venom, which comprises the following steps:
(1) preparation of control solutions
Accurately weighing appropriate amount of pseudo-xenobufotalin, gamabufotalin, bufotalin, bufalin, telocinobufagin, bufotalin, lipobufotalin, south americanum bufotalin, bufalin, lipobufotalin, and cinobufagin reference substance powder, respectively adding methanol and dissolving to obtain reference substance solution. Precisely sucking 0.5mL of each of 11 reference substance solutions, placing in a 10mL volumetric flask, adding methanol to a constant volume, and preparing into a mixed reference substance solution;
(2) preparation of test solution
0.1g of venenum Bufonis powder was weighed out precisely, placed in a 15mL centrifuge tube, and 10mL of methanol was added. Weighing, and ultrasonic extracting for 1 h. And (4) after the ultrasonic treatment is finished, standing the solution to room temperature, and complementing methanol to lose weight. Centrifuging, filtering the filtrate with 0.22 μm filter membrane to obtain sample solution;
(3) respectively measuring the reference substance solution in the step (1) and the test substance solution in the step (2) by adopting HPLC (high performance liquid chromatography), and respectively obtaining the liquid chromatographs of the test substance solution and the reference substance solution;
(4) and (4) introducing the liquid chromatogram obtained in the step (3) into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for analysis, and obtaining HPLC fingerprints of 11 bufogenin components.
The HPLC chromatographic conditions in the step (3) are as follows: gradient elution was performed using an Agilent InfinityLab Poroshell 120 EC-C18 (3.0X 150mm 2.7-Micron, 1000bar) column, mobile phase A being water (containing 0.3% acetic acid), mobile phase B being acetonitrile, wavelength 296nm, flow rate 0.5mL/min, column temperature 30 ℃ and sample loading 5. mu.L.
The gradient elution procedure is as follows:
| time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0～15 | 80→72 | 20→28 |
| 15～30 | 72→71 | 28→29 |
| 30～35 | 71→64 | 29→36 |
| 35～45 | 64→62 | 36→38 |
The HPLC fingerprint establishing method of 11 bufogenin components in the toad venom can be used for toad venom quality evaluation, and fingerprint data obtained in the step (4) is processed by similarity analysis, principal component analysis or clustering analysis, so that the toad venom quality evaluation is carried out.
The invention has the following beneficial effects:
(1) based on the properties of active ingredients in the toad venom and the characteristics of HPLC, single-factor investigation of detection wavelength, chromatographic column, mobile phase, flow rate, column temperature and sample injection amount is carried out, and finally the HPLC detection method for detecting 11 bufogenin active ingredients in the toad venom is obtained through optimization. The method has the advantages of low instrument cost, simple operation, good repeatability, high sensitivity and separation efficiency, is suitable for detecting 11 bufogenin components in the toad venom, and can provide a methodological basis for the subsequent quality control of the toad venom medicinal materials.
(2) According to the invention, a toad venom HPLC fingerprint spectrum is established, and similarity evaluation is carried out on 12 batches of toad venom medicinal material chromatographic peaks, and the result shows that the similarity of toad venom produced in Shandong is greater than 0.9, which indicates that the similarity of characteristic components of the toad venom of different batches is good, but the similarities of the toad venom of S11 and S12 batches are 0.744 and 0.731 respectively, which indicates that the difference of effective components of the toad venom produced in Shandong in 10 batches and the toad venom of other two batches is obvious. The same results were obtained in the principal component analysis and the cluster analysis of 12 batches of the toad venom samples. Therefore, the difference of the quality of the toad venom in different producing areas is revealed, and the fingerprint of the traditional Chinese medicine can be used for revealing the quality difference of the toad venom.
Drawings
FIG. 1 shows the separation results of 11 bufogenin components by different chromatographic columns;
FIG. 2 shows the results of the separation of 11 bufogenin-like components at different flow rates;
FIG. 3 shows the separation of 11 bufogenin-like components at different flow rates;
FIG. 4 shows the separation results of 11 bufogenin components at different column temperatures;
FIG. 5 shows the results of the separation of 11 bufogenin-like components with different sample volumes;
FIG. 6 is HPLC overlay fingerprint of 12 batches of Bufonis venenum samples;
FIG. 7 is HPLC control fingerprint of Bufonis venenum sample;
FIG. 8 is a clustering dendrogram of 12 batches of Bufonis venenum samples;
FIG. 9 is a principal component analysis score chart;
FIG. 10 is a loading chart of principal component analysis of 11 common components;
FIG. 11 is a principal component analysis lithotripsy graph;
FIG. 12 shows the HPLC assay results of the mixed control solution (A) and the toad venom sample solution (B), in which 1-pseudoxenobufalin; 2-gamabufotalin; 3-a xenopus toxicant; 4-bufotalidine; 5-telocinobufagin; 6-bufotalin; 7-lipobufogenin; 8-south American toad venom essence; 9-bufalin; 10-resibufogenin; 11-cinobufagin.
Detailed Description
EXAMPLE 1 instruments, drugs and reagents used in the invention
1.1 Instrument
Agilent 1260 high performance liquid chromatograph (Agilent, USA); sorvall ST 8R centrifuge (Ostred division, thermo-electric Experimental facilities, Inc.); KQ-500DE model digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); ME204/02 electronic balance (Mettler-Torledo instruments, Inc.).
1.2 drugs and reagents
Comparison products: pseudoxenobufotalin (lot number Y18O9Y72597), gamabufotalin (lot number P17O9F72594), bufotalin (lot number Y03D9Y76566), bufotalin (lot number P14J11S118458), xenobufotalin (lot number Y13A9Y67875), bufotalin (lot number P28M10F84299), lipobufotalin (lot number P14J11S118459), nanmei bufotalin (lot number C25F10G81502), bufotalin (lot number P27F10F81703), cinobufagin (lot number W27M10Z84297), lipobufotalin (lot number C30S8G 45143). The 11 reference substances are purchased from Shanghai leaf Biotech company and have mass fractions of more than 98%. Methanol (chromatographic grade), acetonitrile (chromatographic grade), acetic acid (chromatographic grade) were purchased from siemer feishel technologies ltd. Purified water (Jinan Wa Ha beverage Co., Ltd.).
1.3 samples
The toad venom samples used in the present invention are detailed in table 1.
TABLE 1 Bufonis venenum sample information
Example 2 HPLC fingerprint establishing method and fingerprint research for 11 bufogenin components in Bufonis venenum
2.1 preparation of control solutions
Accurately weighing appropriate amount of pseudo-xenobufotalin, gamabufotalin, bufotalin, bufalin, telocinobufagin, bufotalin, lipobufotalin, south americanum bufotalin, bufalin, lipobufotalin, and cinobufagin reference substance powder, respectively adding methanol and dissolving to obtain reference substance solution. Precisely sucking 0.5mL of each of 11 reference solutions, placing in a 10mL volumetric flask, adding methanol to a constant volume, and preparing into a mixed reference solution.
2.2 preparation of test solutions
0.1g of venenum Bufonis powder was weighed out precisely, placed in a 15mL centrifuge tube, and 10mL of methanol was added. Weighing, and ultrasonic extracting for 1 h. And (4) after the ultrasonic treatment is finished, standing the solution to room temperature, and complementing methanol to lose weight. Centrifuging, filtering the filtrate with 0.22 μm filter membrane to obtain test solution.
2.3 chromatographic conditions
2.3.1 selection of detection wavelength
According to the invention, full-wavelength scanning is carried out on the components of the toad venom within the range of 190-400 nm, and the result shows that most of the components of the toad venom have maximum absorption within the range of 290-310 nm. Combining the ultraviolet scanning spectrum results of 11 bufogenin components, referring to the pharmacopoeia of the people's republic of China (2020 edition) and related documents of quality control of the toad venom, giving consideration to the detection requirements of the 11 bufogenin components in the toad venom, and finally selecting 296nm as the detection wavelength of the invention.
2.3.2 selection of chromatography columns
According to the invention, three chromatographic columns of Agilent InfinityLab Poroshell 120 EC-C18 (3.0X 150mm 2.7-Micron), Agilent ZORBAX SB-C18 (4.6X 250mm,5-Micron), and Agilent ZORBAX Eclipse XDB-C18 (4.6X 250mm,5-Micron) are examined and compared according to the difference of column length and packing. Finally, an Agilent InfinityLab Poroshell 120 EC-C18 chromatographic column is selected, has better separation degree and shorter separation time, and can be used for content determination of 11 bufogenin components in the invention.
2.3.3 selection of the Mobile phase
The invention considers three mobile phases of acetonitrile-pure water, acetonitrile-water (containing 0.3 percent of acetic acid) and methanol-water (containing 0.3 percent of acetic acid). As shown in FIG. 2, the results of the analyses of acetonitrile and methanol as mobile phases are significantly different from each other, and when the organic phase is acetonitrile, the separation of the components is good. The result also shows that whether the acid is added has no obvious influence on the detection result of the 11 components, but the problems of mobile phase deterioration, baseline fluctuation and the like caused by taking pure water as the mobile phase can be relieved after the proper amount of acid is added. Therefore, acetonitrile-water (containing 0.3% acetic acid) was finally selected as the mobile phase.
2.3.4 selection of flow Rate
The flow rate of the present invention to this column (0.2 mL. min)-1、0.5mL·min-1、0.8mL·min-1) Optimization is performed. When the flow rate is 0.8 mL/min-1Meanwhile, the service life of the chromatographic column may be affected due to too high pressure of the chromatographic column, and as shown in fig. 3, the separation effect of bufotalin and resibufogenin is not ideal; when the flow rate is 0.2 mL/min-1In this case, it takes a long time to separate all the components. Therefore, the present invention finally determines the flow rate to be 0.5mL/min-1。
2.3.5 selection of column temperature
The invention also considers the influence of the column temperature (25 ℃, 30 ℃, 35 ℃ and 40 ℃) on the separation effect of the 11 bufogenin components in the toad venom. As shown in FIG. 4, at a column temperature of 25 ℃ the peak response values of the individual components are too small to be used for quantitative determination of the components; when the column temperature is 35 ℃, the separation effect of the bufotalin and the resibufotoxin is worse than that of 30 ℃; when the column temperature is 40 ℃, chromatographic peaks of the bufotalin and the resibufotoxin cannot be completely separated; when the column temperature is 30 ℃, the chromatographic peak shapes of the 11 bufogenin components are good, and the separation effect is optimal.
2.3.6 selection of sample size
The present invention compares the effect of sample loading (3. mu.L, 5. mu.L, 10. mu.L) on the assay results without changing other chromatographic conditions. As shown in FIG. 5, when the amount of the sample was 3. mu.L, the peak area of each component was small and the error was large; when the sample size was 10. mu.L, the first two peaks exhibited shoulders, which may be due to the shorter length and smaller inner diameter of the Agilent InfinityLab Poroshell 120 EC-C18 column, resulting in overloading of the sample. Therefore, the present invention finally determines the sample amount to be 5 μ L.
2.3.7 chromatographic conditions
The chromatographic conditions of the invention are specifically as follows: by Agilent InfinityLab Poroshell 120 EC-C18 (3.0X 150mm 2.7-Micron, 1000bar) column was subjected to gradient elution. The mobile phase was water (containing 0.3% acetic acid) (A) -acetonitrile (B), wavelength 296nm, flow rate 0.5 mL. min-1The column temperature was 30 ℃ and the amount of sample was 5. mu.L. The gradient elution procedure is shown in table 2.
TABLE 2 gradient elution procedure
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0～15 | 80→72 | 20→28 |
| 15～30 | 72→71 | 28→29 |
| 30～35 | 71→64 | 29→36 |
| 35～45 | 64→62 | 36→38 |
2.4 fingerprint Studies
2.4.1 creation of fingerprint and identification of chromatographic peaks
Taking S1-S12 batches of toad venom samples, preparing a test solution according to the method under item 2.2, performing HPLC analysis according to the chromatographic condition under item 2.3, and introducing the obtained chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) to obtain 12 batches of toad venom fingerprint common modes and a reference chromatogram (figure 6). 26 common peaks are marked in the contrast finger print of 12 batches of venenum bufonis medicinal materials, 11 chromatographic peaks (figure 7) are identified in total by consulting literature data and comparing with the chromatogram of a mixed contrast solution, wherein the chromatographic peaks are respectively pseudo-xenobufotalin (peak 2), gamabufotalin (peak 3), bufotalin (peak 7), bufotalin (peak 8), telocinobufagin (peak 17), bufotalin (peak 19), lipobufotalin (peak 20), south americanum bufotalin (peak 22), bufalin (peak 24), lipobufogenin (peak 25) and bufogenin (peak 26).
2.4.2 Similarity Analysis (SA)
The fingerprint of 12 batches of the toad venom medicinal materials is subjected to correlation coefficient calculation by using a traditional Chinese medicine fingerprint similarity evaluation system (2012 edition) and reference spectra, the similarity and the difference of the samples are classified, and the similarity of the samples is evaluated by using the correlation coefficients. The fingerprint spectrum of 12 batches of toad venom is compared with the reference spectrum, and the result shows that the similarity of the toad venom produced in Shandong is 0.986-1.000, and the similarities of the two batches of the toad venom, S11 and S12, are 0.744 and 0.731 (see table 3), which shows that the toad venom produced in Shandong in the research has better quality and relatively stable quality.
HPLC fingerprint similarity of Table 312 sets of Bufonis venenum samples
Example 3 chemical Pattern analysis
3.1 Cluster analysis (HCA)
The invention uses SPSS 22.0 software to cluster by using the content of 11 compounds in 12 batches of medicinal materials as variables. Clustering analysis can distinguish different batches of toad venom samples, and the results are shown in fig. 8, wherein when the classification distance is 15 or 20, one is S1-S10, and the other is S11 and S12.
3.2 principal Components analysis
According to the method, the relative peak areas of 11 common peaks in the toad venom are used as indexes, the main component analysis is carried out on the contents of 11 components in 12 batches of the toad venom, a score map (figure 9) and a load map (figure 10) of the 11 components in 12 batches of the toad venom samples are obtained, S1-S10 are classified into one type, S11 and S12 are classified into one type, and the clustering analysis result is consistent with that of the two types. The result shows that the fingerprint spectrums of the ingredients of the Shandong toad venom and the S11 and S12 toad venom in two batches are obviously different, and the toad venom samples in different batches are different. Because the weighted value obtained by calculating the point far away from the origin in the load graph is the largest, the Chinese bufogenin, bufotalin and bufogenin of the invention play a great role in sample classification.
The invention comprehensively considers three factors of the steepness degree (figure 11) among all components of the lithotripsy, whether the characteristic value of the principal component analysis is more than 1 and the variance contribution rate (table 4) to determine the optimal number of the principal components. The result shows that the ingredients with large loading capacity corresponding to the main ingredient 1 of the toad venom comprise cinobufagin, bufotadin and bufalin, which shows that the main ingredient 1 is greatly influenced by the three ingredients and provides a basis for quantitative evaluation of multiple ingredients; regarding the main component 2, the components with large loading amount include south America toad venom essence, lipo toad venom essence and telocinobufagin; it is shown that the main component 2 is greatly affected by these three components. The cumulative variance contribution rate of 5 main components extracted from the venenum bufonis medicinal materials is 98.660%, and the rubble chart shows that the straight line of the components at the top 5 is steep, which is helpful for explaining variables and can integrate most information of the venenum bufonis components. According to the two main components extracted by the main component analysis, the invention shows that the quality of the S1 batch of toad venom is the best in 12 batches of toad venom samples, and the quality of the S11 batch of toad venom and the S12 batch of toad venom are poor, which indicates that the quality of the 10 batches of Shandong toad venom is better in the HPLC fingerprint and analysis established by the invention.
TABLE 4 principal component analysis eigenvalues and variance contribution rates
Example 4 methodology examination
4.1 specialization examination
Precisely measuring the sample solution (S2 batch) under the item "2.2" for content determination under the chromatographic condition under the item "2.3", and combining the chromatographic results of the mixed reference solution under the item "2.1", as shown in FIG. 12, obtaining the characteristic peaks of the 11 bufogenin active ingredients, wherein the separation degree of the target ingredient from impurities is good without interference.
4.2 Linear relationship investigation
Diluting the control solution under the term of "2.1" to 5, 10, 50, 100, 200, 500 μ g/mL-1The control solution of (4). And (3) performing content measurement according to the chromatographic condition under the item of 2.3, finally drawing a standard curve by taking the mass concentration of each component as an abscissa (X) and the peak area as an ordinate (Y), and determining a regression equation.
Regression equation and linear range of table 511 toad venom complex base class components
4.3 precision test
Precisely measuring 100 μ L of the test solution of Bufonis venenum sample (S2 batch) of the same batch under item "2.2", and performing sample injection measurement for 6 times under chromatographic condition under item "2.3". As shown in Table 6, the RSD values of the finally calculated relative peak areas were all less than 1.34%, indicating that the precision of the instrument was good.
Table 611 precision investigation results of ingredients of bufogenin ligand class
2.6.4 repeatability test
Precisely weighing 6 parts of the same batch of venenum bufonis samples (S3 batch), respectively preparing 6 parts of test solution in parallel according to the method under item 2.2, and performing content determination according to the chromatographic condition under item 2.3. As shown in Table 7, the RSD values of the relative peak areas of the 11 active ingredients are all less than 4.70%, indicating that the method has good repeatability.
Table 711 repeated investigation results of ingredients of complex class of bufogenin
4.5 stability test
The content of the same test solution (batch S3) is respectively measured at 0h, 2h, 4h, 8h, 12h and 24h, and as shown in Table 8, the RSD values of the relative peak areas of the 11 components are less than 2.89%, thus verifying that the test solution has good stability within 24 h.
Table 811 ingredient stability test results for bufotoxin ligand class
4.6 sample recovery test
The 12 batches of the toad venom samples of known content were weighed out in 9 portions, each about 0.1 g. The venenum bufonis active ingredient was extracted according to the method under item "2.2", and the content was measured according to the chromatographic conditions under item "2.3", and the recovery rate was calculated, and the results are shown in table 9.
Table 911 bufogenin components sample recovery rate investigation results (n ═ 9)
4.7 assay of sample
Taking 12 batches of toad venom samples, and respectively carrying out three times of content measurement in parallel. The results of measuring the contents of the 12 batches of the toad venom samples are shown in table 10. The inventor finds that the total content of bufalin, cinobufagin and resibufogenin in 10 batches of Shandong toad venom samples meets the standard of 'Chinese pharmacopoeia' 2020 edition, and other two batches of unknown toad venom samples do not meet the standard.
TABLE 1012 determination of 11 active ingredient content (mg/g) in Lou Bufonis venenum
Claims (3)
1. The HPLC fingerprint spectrum establishment method of 11 bufogenin components in the toad venom is characterized by comprising the following steps:
(1) preparation of control solutions
Accurately weighing appropriate amount of pseudo-xenobufagin, gamabufotalin, bufalin, telocinobufagin, bufalin, lipobufalin, south American bufalin, lipobufalin genin, and cinobufagin reference substance powder, respectively adding methanol and dissolving to obtain reference substance solution; precisely absorbing 11 reference substance solutions, placing in a volumetric flask, adding methanol to a constant volume, and preparing into a mixed reference substance solution;
(2) preparation of test solution
Precisely weighing venenum bufonis powder, placing in a centrifuge tube, adding methanol, weighing, performing ultrasonic extraction, finishing ultrasonic treatment, standing the solution to room temperature, complementing methanol to zero gravity, centrifuging, taking subsequent filtrate, and filtering with a membrane to obtain a test solution;
(3) respectively measuring the reference substance solution in the step (1) and the test substance solution in the step (2) by adopting HPLC (high performance liquid chromatography), and respectively obtaining the liquid chromatographs of the test substance solution and the reference substance solution;
(4) introducing the liquid chromatogram obtained in the step (3) into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for analysis to obtain HPLC fingerprints of 11 bufogenin components;
the HPLC chromatographic conditions in the step (3) are as follows: gradient elution is carried out by adopting an Agilent InfinityLab Poroshell 120 EC-C18 chromatographic column, the specification of the chromatographic column is 3.0 multiplied by 150mm 2.7-Micron, 1000 bar; the mobile phase A is water containing 0.3% acetic acid, the mobile phase B is acetonitrile, the wavelength is 296nm, the flow rate is 0.5mL/min, the column temperature is 30 ℃, and the sample injection amount is 5 mu L;
the gradient elution procedure is as follows:
2. the use of the HPLC fingerprint spectrum establishment method of 11 bufogenin components in venenum bufonis of claim 1 in quality evaluation of venenum bufonis.
3. The use of the HPLC fingerprint establishment method for 11 bufogenin components in Bufonis venenum according to claim 2 in quality evaluation of Bufonis venenum, wherein the fingerprint data obtained in step (4) is processed by similarity analysis, principal component analysis or clustering analysis, thereby performing quality evaluation of Bufonis venenum.
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