CN114062525A - Radix astragali-bone capsule fingerprint detection method, control fingerprint and application - Google Patents

Radix astragali-bone capsule fingerprint detection method, control fingerprint and application Download PDF

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CN114062525A
CN114062525A CN202010771890.1A CN202010771890A CN114062525A CN 114062525 A CN114062525 A CN 114062525A CN 202010771890 A CN202010771890 A CN 202010771890A CN 114062525 A CN114062525 A CN 114062525A
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fingerprint
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关斌
阳丽华
许舒瑜
南淑华
赖志成
陈晓琳
郑珊珊
许玉珍
肖惠琳
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Xiamen Traditional Chinese Medicine Co ltd
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Abstract

The invention relates to a radix astragali-bone capsule fingerprint detection method, a reference fingerprint and application. The method adopts a high performance liquid chromatograph equipped with an ultraviolet detector to carry out detection, and the chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX SB-C18 chromatographic column with the thickness of 250mm × 4.6mm and the thickness of 5 μm, and the column temperature is 30 ℃; the flow rate is 1 ml/min; the detection wavelength is 270 nm; the sample amount is 10 mul; mobile phase: methanol (a) -acetonitrile (B) -0.1% aqueous phosphoric acid (C); gradient elution. The obtained Qigu capsule can be used for quality control of Qigu capsule by referring to fingerprint.

Description

Radix astragali-bone capsule fingerprint detection method, control fingerprint and application
Technical Field
The invention belongs to the field of traditional Chinese medicine analysis, and relates to a liquid chromatography method for detecting a Qigu capsule fingerprint, a method for establishing a contrast fingerprint containing the method, a Qigu capsule contrast fingerprint and application.
Background
The radix astragali and bone capsule comprises herba Epimedii, radix Polygoni Multiflori Preparata, radix astragali, herba Dendrobii, Cistanchis herba, rhizoma Drynariae, and flos Chrysanthemi. Has effects of nourishing liver and kidney, strengthening tendons and bones. Can be used for treating female postmenopausal osteoporosis with syndrome of deficiency of liver and kidney, manifested by soreness of waist and knees, asthenia, lumbago, backache, difficulty in walking, and inability to bear heavy weight.
In recent years, as higher requirements on drug quality control are provided by regulatory departments, manufacturers are required to strictly control the stability among product batches, so that the adoption of an effective quality control method is particularly important.
The fingerprint spectrum technology of traditional Chinese medicine is increasingly used for quality control of traditional Chinese medicine due to the characteristics of comprehensively marking the characteristics and proportion of the main chemical components of the traditional Chinese medicine. The chromatography is one of analysis methods which are developed rapidly and widely applied in the field of analytical chemistry, and is also the most basic technology of the traditional Chinese medicine fingerprint. Common Chromatography methods include thin layer Chromatography, Liquid Chromatography, gas Chromatography and capillary electrophoresis, wherein High Performance Liquid Chromatography (HPLC) has the advantages of wide application range, High analysis speed, High sensitivity and the like, and is the most widely applied method for researching traditional Chinese medicine fingerprints at present.
The main active ingredients in the astragalus-bone capsule comprise echinacoside, calycosin glucoside, 2,3,5, 4-tetrahydroxystilbene glucoside, naringin, epimedin A, epimedin B, epimedin C, icariin, icarisid I, baohuoside I, emodin and the like.
At present, few researches on establishing a liquid chromatography method for detecting the fingerprint of the Qigu capsule and adopting the method for quality control are available.
Disclosure of Invention
In order to more comprehensively and effectively control the quality of the stilbene bone capsule, the inventor adopts an HPLC method and researches conditions such as a mobile phase, a detection wavelength, a chromatographic column, a column temperature, a gradient condition, a flow rate, a sample injection amount and the like to obtain a liquid chromatography method for detecting the stilbene bone capsule, and simultaneously researches an extraction solvent, an extraction mode, extraction time, a solvent amount and the like to obtain a method for preparing a test solution, thereby obtaining a method for establishing the fingerprint of the stilbene bone capsule, and obtaining a reference fingerprint of the stilbene bone capsule by the method, wherein the reference fingerprint can be used for controlling the quality of the stilbene bone capsule, thereby completing the invention.
The invention provides a liquid chromatography method for detecting the fingerprint of Qigu capsules, which adopts a high performance liquid chromatograph equipped with an ultraviolet detector to detect, wherein the chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX SB-C18 column, 250mm 4.6mm, 5 μm,
column temperature: 30 deg.C
Flow rate: 1 ml/min;
detection wavelength: 270 nm;
sample introduction amount: 10 μ l
Mobile phase: methanol (A) -acetonitrile (B) -0.1% phosphoric acid aqueous solution (C)
Gradient elution conditions:
t/min methanol% Acetonitrile% 0.1% phosphoric acid aqueous solution%
0 4 1 95
10 11 7 82
33 11 19 70
47 16 24 60
57 25 34 41
67 56 34 10
77 4 1 95
。
In the present invention, the percentages in the mobile phase and gradient elution are volume percentages, for example, 0.1% means 0.1 volume%.
In the liquid chromatography method for detecting the fingerprint of the Qigu capsule, the Qigu capsule test solution for detection can be prepared as follows: extracting the content of the astragalus-bone capsule with methanol at a liquid-solid ratio (volume/mass, ml/g) of 50-200 times. The extraction method can be ultrasonic treatment, reflux extraction and the like, and ultrasonic extraction is preferred. The extraction time may be 15 minutes or more, for example, 15 to 60 minutes, preferably 30 to 45 minutes.
Specifically, the test solution of the stilbene bone capsule can be prepared as follows: weighing 0.5g of radix astragali bone capsule content, precisely weighing, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, supplementing loss mass with methanol, and filtering with 0.45 μm microporous membrane to obtain test solution.
In another aspect, the present invention provides a method for establishing a astragalus bone capsule control fingerprint, which comprises:
(1) preparing a test solution:
weighing 0.5g of radix astragali bone capsule content, precisely weighing, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, supplementing loss mass with methanol, and filtering with 0.45 μm microporous membrane;
(2) preparing a reference substance solution:
respectively taking a proper amount of echinacoside, calycosin glucoside, 2,3,5, 4-tetrahydroxystilbene glucoside, naringin, epimedin A, epimedin B, epimedin C, icariin, icarisid I, baohuoside I and emodin, precisely weighing, and adding methanol to prepare a mixed solution containing 18.55 mu g of echinacoside, 25.22 mu g of calycosin glucoside, 30.95 mu g of 2,3,5, 4-tetrahydroxystilbene glucoside, 26.89 mu g of naringin, 25.75 mu g of epimedin A, 20.00 mu g of epimedin B, 22.48 mu g of epimedin C, 18.92 mu g of icariin, 26.60 mu g of icarisid I, 22.70 mu g of baohuoside I and 16.07 mu g of emodin in each 1 ml;
(3) preparation of negative sample solution:
taking 0.5g of negative sample powder lacking herba Epimedii, radix Polygoni Multiflori Preparata, radix astragali, herba Dendrobii, Cistanchis herba, rhizoma Drynariae or flos Chrysanthemi, precisely weighing, precisely adding 50ml of methanol, weighing, performing ultrasound for 30min, supplementing the loss mass with methanol, filtering with 0.45 μm microporous membrane, and preparing each negative sample solution;
(4) preparing a medicinal material solution:
respectively extracting herba Epimedii, radix Polygoni Multiflori Preparata, radix astragali, herba Dendrobii, Cistanchis herba, rhizoma Drynariae, and flos Chrysanthemi with water under reflux, concentrating, filtering with 0.45 μm microporous membrane, and making into solution;
(5) and (4) HPLC detection:
respectively and precisely absorbing 10 mu l of each of the test solution, the reference solution, the negative sample solution and the medicinal material solution of the (1), (2), (3) and (4), and detecting by using the liquid chromatography method for detecting the fingerprint of the stilbene bone capsule to obtain a test sample fingerprint chromatogram, a reference fingerprint chromatogram, a negative sample fingerprint chromatogram and a medicinal material fingerprint chromatogram;
(6) generating a standard fingerprint spectrum:
generating a comparison fingerprint spectrum based on common peaks in fingerprint chromatograms of N batches of test samples, wherein chromatographic peaks with good separation degree of main components in the Qigu capsules are selected as characteristic peaks, and 15 common peaks are determined, wherein N is more than 10, and is preferably 10-30;
(7) identification and attribution of common peaks:
and (4) attributing and identifying common peaks in the Qigu capsule fingerprint.
In the above method, the method for generating the control fingerprint based on the sample fingerprint chromatogram is not particularly limited, and a conventional method in the art may be employed. For example, the measured fingerprint chromatogram of the stilbene bone capsule test sample can be introduced into a similarity evaluation system (2012 edition) of traditional Chinese medicine chromatogram fingerprints issued by the national pharmacopoeia committee, one of the chromatogram chromatograms of the test sample is set as a reference spectrum, a standard spectrum generation method is a median method for example, and the reference fingerprint of the stilbene bone capsule is established by adopting a multipoint correction method for example.
In the method, identification and attribution of the common peaks can be completed by comparing with chromatograms of the negative sample, the single medicinal material and the reference substance solution.
Another aspect of the present invention provides a astragalus bone capsule control fingerprint, which is substantially as shown in fig. 6, and comprises 15 characteristic peaks.
Comparing with chromatogram of negative sample, single medicinal material and control solution, wherein 7, 8, 9, 10, 11, 12, 13, 14 belong to herba Epimedii in 15 characteristic peaks; peak 4, 15 belongs to Polygonum multiflorum; peak 5, 6 belong to drynaria; peak 3 belongs to Astragalus membranaceus; peak 2 belongs to Cistanchis herba. In addition, a chemical composition of 11 peaks was identified, peak 2: echinacoside; peak 3: calycosin glucoside; peak 4: 2,3,5, 4-tetrahydroxystilbene glucoside; peak 6: naringin; peak 7: epimedin A; peak 8: epimedin B; peak 9: epimedin C; peak 10: icariin; peak 11: icariside I; peak 14: baohuoside I; peak 15: emodin is added.
In an embodiment, the astragalus bone capsule contrast fingerprint spectrum is obtained by the method for establishing the astragalus bone capsule contrast fingerprint spectrum according to the invention.
In another aspect, the present invention provides a quality control method of a Qigu capsule, which comprises the following steps:
(1) preparing a test solution:
taking 0.5g of the content of the stilbene bone capsule sample to be detected, precisely weighing, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, complementing loss mass with methanol, and filtering through a 0.45 mu m microporous membrane;
(2) chromatographic conditions are as follows:
adopting the chromatographic conditions in the liquid chromatographic method for detecting the fingerprint of the Qigu capsule;
(3) and (3) determination:
precisely sucking 10 mu l of the solution (1), injecting into a liquid chromatograph, and measuring to obtain a fingerprint of the stilbene bone capsule sample to be detected;
(4) and (4) qualification judgment:
and calculating the similarity between the fingerprint of the stilbene bone capsule sample to be detected and the reference fingerprint, wherein the similarity is more than or equal to 0.8, and obtaining a qualified product.
The similarity can be obtained by introducing the fingerprint of the stilbene bone capsule sample to be detected and the reference fingerprint of the stilbene bone capsule into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system issued by the State pharmacopoeia Committee for comparison, so as to calculate the similarity between the sample fingerprint and the reference fingerprint, wherein the similarity is more than or equal to 0.8, preferably more than 0.9, namely the qualified product is obtained, and otherwise, the unqualified product is obtained.
As proved by experimental results, the chromatogram obtained by the method for detecting the finger print of the astragalus-bone capsule has the advantages of more peak number, high sensitivity, stable base line, low noise, sharp response, large response value and good separation degree. In addition, the blank solvent has no interference on the test of the test solution, and has good specificity. The method has good precision, stability and reproducibility.
The present invention has been described in detail hereinabove, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. Furthermore, there is no intention to be bound by any theory presented in the preceding prior art or the summary or the following examples.
Unless expressly stated otherwise, a numerical range throughout this specification includes any sub-range therein and any numerical value incremented by the smallest sub-unit within a given value. Unless expressly stated otherwise, numerical values throughout this specification represent approximate measures or limitations to the extent that such deviations from the given values, as well as embodiments having approximately the stated values and having the exact values stated, are included. Other than in the operating examples provided at the end of the detailed description, all numbers expressing quantities or conditions of parameters (e.g., quantities or conditions) used in the specification (including the appended claims) are to be understood as being modified in all instances by the term "about" whether or not "about" actually appears before the number. "about" means that the numerical value so stated is allowed to be somewhat imprecise (with some approach to exactness in that value; about or reasonably close to that value; approximately). As used herein, "about" refers to at least variations that can be produced by ordinary methods of measuring and using such parameters, provided that the imprecision provided by "about" is not otherwise understood in the art with this ordinary meaning. For example, "about" can include less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.1% variation, and in some aspects, less than or equal to 0.01% variation.
The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein. Various modifications, changes, or substitutions may be made to the technical solution of the present invention without departing from the scope of the technical idea and technical spirit of the present invention, and the modified, changed, or substituted technical solution is still included in the scope of the present invention.
It will be understood that the words or terms used in the specification and claims should not be construed as meaning defined in commonly used dictionaries. It will be further understood that the terms or terms should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the technical spirit of the present invention, based on the principle that the inventor can appropriately define the meaning of the terms or terms to best explain the present invention.
Drawings
Fig. 1 shows a liquid chromatogram for detecting a stilbene bone capsule test solution and a blank solvent according to the chromatographic conditions of the present invention, a: the test solution of the Qigu capsule; b: a blank solvent.
FIG. 2 shows a liquid chromatogram obtained by examining the precision of a sample solution of the same Qigu capsule according to the method of the present invention.
FIG. 3 shows a liquid chromatogram obtained by performing a stability test on a test solution of the same portion of Qigu capsules according to the method of the present invention.
FIG. 4 shows a liquid chromatogram obtained from a repeated experiment of the same batch of Qigu capsule powder according to the method of the present invention.
Fig. 5 shows a liquid chromatogram obtained by fingerprint analysis of 21 batches of astragalus bone capsules according to the method of the present invention.
Fig. 6 shows the contrast fingerprint of Qigu capsules established by the method of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the drawings and technical solutions, but the present invention is not limited thereto.
1. Instrument and reagent
The instrument comprises the following steps:
ME1002E electronic balance (Mettler-Tollido instruments (Shanghai) Co., Ltd.), HH-4 digital display constant temperature water bath (Changzhou China electric appliances Co., Ltd.), KQ-600DE numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.), Waters e2695 high performance liquid chromatograph (Watts Co., Ltd.).
Experiment medication:
qigu capsules (batch No. 180801, 180802, 180901, 180902, 180903, 181101, 181102, 181202, 190201, 190202, 190203, 190402, 190403, 190501, 190502, 190503, 190602, 190603, 190701, 190702, 190703, manufacturer: Xiamen Chinese medicine factory Co., Ltd.)
The epimedium deficiency negative sample, the polygonum multiflorum deficiency negative sample, the astragalus root deficiency negative sample, the dendrobium stem deficiency negative sample, the cistanche deficiency negative sample, the rhizoma drynariae deficiency negative sample and the chrysanthemum flower deficiency negative sample are prepared by laboratories according to preparation related processes.
Herba Epimedii (batch No. 171001, supplier: Hebei Huadu pharmaceutical Co., Ltd.), Polygoni Multiflori radix (batch No. H2019031801, supplier: Shanghai Huayu pharmaceutical Co., Ltd.), radix astragali (batch No. 190301-1, supplier: Gansu Ruilong pharmaceutical Co., Ltd.), herba Dendrobii (batch No. 171201, supplier: Xinhua Chinese medicinal material Co., Ltd., Anguo City), Cistanchis herba (batch No. 181201, supplier: Gansu Ruilong pharmaceutical Co., Ltd.), rhizoma Drynariae (batch No. 170501, supplier: Shanghai Huayu pharmaceutical Co., Ltd.), and flos Chrysanthemi (batch No. 180701, supplier: Xiamen pian Huang Hongyun medicine Co., Ltd.).
Comparison products:
echinacoside (batch No. 111670-,
calycosin glucoside (batch No. 111920-201606, 97.6%, China institute for testing food and drug),
2,3,5, 4-Tetrahydroxystilbene glucoside (batch No. must-18051611, 99.44%, Dowmastt Biotech Co., Ltd.),
naringin (batch No. 110722 and 201815, 91.7%, China institute for testing food and drug),
epimedin A (batch No. must-18072304, 98.13%, Dowman Techni Co., Ltd.),
epimedin B (batch No. must-18071403, 99.23%, Dowman Techni Co., Ltd.),
epimedin C (batch No. 111780-201804, 92.6%, China institute for food and drug testing),
icariin (batch No. 110737-,
icariside I (batch No. must-18102120, 97.78%, Doctorite Biotech Co., Ltd.),
baohuoside I (batch No. must-18042203, 99.58%, Doudamant Biotech Co., Ltd.),
emodin (batch No. 110756-201512, 98.7%, China institute for food and drug testing).
2. Liquid phase conditions
By researching chromatographic column, column temperature, flow rate, detection wavelength, sample injection quantity, mobile phase, gradient condition and the like, the following chromatographic conditions are obtained:
a chromatographic column: agilent ZORBAX SB-C18 column (250mm 4.6mm, 5 μm),
column temperature: 30 deg.C
Flow rate: 1 ml/min;
detection wavelength: 270 nm;
sample introduction amount: 10 μ l
Mobile phase: methanol (A) -acetonitrile (B) -0.1% phosphoric acid aqueous solution (C)
Gradient elution:
TABLE 1 gradient elution conditions
Figure BDA0002616946840000061
3. Solution preparation
Preparing a test solution:
weighing 0.5g of radix astragali bone capsule content, precisely weighing, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, supplementing loss mass with methanol, filtering with 0.45 μm microporous membrane, and sampling and analyzing the filtrate.
Preparing a reference substance solution:
taking proper amount of echinacoside, calycosin glucoside, 2,3,5, 4-tetrahydroxystilbene glucoside, naringin, epimedin A, epimedin B, epimedin C, icariin, icariside I, baohuoside I and emodin, precisely weighing, and adding methanol to prepare a mixed solution containing 18.55 mu g of echinacoside, 25.22 mu g of calycosin glucoside, 30.95 mu g of 2,3,5, 4-tetrahydroxystilbene glucoside, 26.89 mu g of naringin, 25.75 mu g of epimedin A, 20.00 mu g of epimedin B, 22.48 mu g of epimedin C, 18.92 mu g of icariin, 26.60 mu g of icariside I, 22.70 mu g of baohuoside I and 16.07 mu g of emodin in each 1 ml.
Preparation of negative sample solution lacking medicinal materials:
taking 0.5g of negative sample powder lacking herba Epimedii, radix Polygoni Multiflori Preparata, radix astragali, herba Dendrobii, Cistanchis herba, rhizoma Drynariae or flos Chrysanthemi, precisely weighing, precisely adding 50ml of methanol, weighing, performing ultrasound for 30min, supplementing the lost mass with methanol, filtering with 0.45 μm microporous membrane, and sampling and analyzing the filtrate.
Preparing a medicinal material solution:
respectively extracting herba Epimedii, radix Polygoni Multiflori Preparata, radix astragali, herba Dendrobii, Cistanchis herba, rhizoma Drynariae, and flos Chrysanthemi with water in laboratory under reflux, concentrating, filtering with 0.45 μm microporous membrane, and sampling and analyzing the filtrate.
4. Establishing an extraction method
The establishment of the extraction method comprises the investigation of the extraction mode, the extraction solvent, the solvent volume and the extraction time.
(1) Examination of extraction method and extraction solvent
Preparing a test solution: taking 0.5g of Qigu capsule powder, precisely weighing 8 parts in total, placing the powder in a conical flask with a plug, precisely adding 50ml of different solvents according to the table 2, processing for 60 minutes, cooling, weighing again, supplementing the lost weight with the corresponding solvent, shaking up, filtering to obtain a test solution, and analyzing the test. The results are shown in Table 3.
TABLE 2 Experimental methods
Figure BDA0002616946840000071
TABLE 3 examination of extraction methods and extraction solvents
Figure BDA0002616946840000081
The results in table 3 show that the icariin content in the methanol ultrasonic and methanol reflux extraction is higher than that in the ethanol ultrasonic and reflux extraction, and the icariin content in the methanol ultrasonic and reflux extraction has no significant difference, so the extraction mode can be ultrasonic treatment or reflux extraction. In view of the convenience of the future operation, methanol ultrasound is preferably selected as the experimental method.
(2) Solvent volume investigation
Different volumes were examined and the results are shown in table 4.
Table 4 table of solvent volume investigation results
Figure BDA0002616946840000082
The result shows that the content of the icariin extracted by the extraction volume of 50ml and 75ml is higher than that of 25 ml and 100ml, and the content of the icariin extracted by the extraction volume of 50ml and 75ml has no obvious difference, so that the icariin can be extracted by methanol at a liquid-solid ratio (volume/mass, ml/g) of 50-200 times. Considering cost savings, an extraction volume of 50ml was chosen.
(3) Extraction time review
The results of different extraction times are shown in Table 5.
TABLE 5 extraction of time survey results table
Figure BDA0002616946840000091
The results show that the extraction time is 30 to 45min, the content of icariin is slightly higher than the extraction time is 15 to 60min, and therefore, the extraction time can be 15 minutes or more, for example, 15 to 60 minutes, preferably 30 to 45 minutes. Considering the convenience of operation in the future, the extraction time is preferably selected to be 30min,
(4) small knot
The determined optimal extraction conditions are as follows: weighing 0.5g of radix astragali bone capsule content, precisely weighing, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, supplementing loss mass with methanol, filtering with 0.45 μm microporous membrane, and sampling filtrate for analysis.
5. Methodology study
(1) Specialization inspection
Respectively sampling blank solvent and radix astragali bone capsule sample solution, respectively injecting sample according to the above liquid phase conditions, and recording chromatogram, the result is shown in figure 1.
The result shows that the blank solvent has no interference to the test of the test solution, and the method has good specificity.
(2) Precision survey
Taking a sample solution of the same Qigu capsule, continuously injecting sample for 6 times according to the above liquid phase conditions, and recording chromatogram, wherein the results are shown in FIG. 2 (precision experiments 1-6 are respectively indicated as S1-S6, and reference chromatogram is indicated as R).
Relative retention times and relative peak areas were calculated for the 15 main chromatographic peaks, using peak number 10 as reference, and the results are shown in tables 6 and 7.
TABLE 6 precision test results (relative Retention time)
Figure BDA0002616946840000101
TABLE 7 precision test results (relative peak area)
Figure BDA0002616946840000102
The experimental results show that the RSD of relative retention time of 15 main chromatographic peaks is between 0.01 and 0.09 percent, the RSD of relative peak area is between 0.09 and 2.29 percent, and the RSD is less than 3.0 percent.
The above experimental results show that the precision of the method according to the invention is good.
(3) Stability test
Sample solutions of the same Qigu capsules are taken, and are injected at 0, 2, 4, 6, 10, 16, 24, 36, 48 and 72h respectively according to the chromatographic conditions, chromatograms are recorded, and the results are shown in FIG. 3 (wherein the experiments of 0, 2, 4, 6, 10, 16, 24, 36, 48 and 72h are respectively indicated as S1-S10 in the figure, and the comparison chromatograms are indicated as R). Relative retention times and relative peak areas were calculated for the 15 main chromatographic peaks, using peak number 10 as reference, and the results are shown in tables 8 and 9.
TABLE 8 stability test results (relative Retention time)
Figure BDA0002616946840000111
TABLE 9 stability test results (relative peak area)
Figure BDA0002616946840000112
The experimental results show that the RSD of relative retention time of 15 main chromatographic peaks is between 0.01 and 0.62 percent, the RSD of relative peak area is between 0.49 and 3.18 percent, and the RSD is less than 5.0 percent. The test article prepared according to the method of the present invention was shown to be substantially stable within 72 hours.
(4) Repeatability test
Precisely weighing 6 parts of the same batch of the astragalus bone capsule powder, preparing 6 parts of the test solution in parallel according to the preparation method of the test solution, respectively carrying out sample injection analysis according to the chromatographic conditions, and recording a chromatogram, wherein the result is shown in figure 4 (wherein the reproducibility experiments 1-6 are respectively indicated as S1-S6, and the reference chromatogram is indicated as R). Relative retention times and relative peak areas were calculated for the 15 main chromatographic peaks, using peak number 10 as reference, and the results are shown in tables 10 and 11.
TABLE 10 reproducibility test results (relative Retention time)
Figure BDA0002616946840000121
TABLE 11 reproducibility test results (relative peak area)
Figure BDA0002616946840000122
Figure BDA0002616946840000131
The experimental results show that the RSD of relative retention time of 15 main chromatographic peaks is between 0.01 and 1.15 percent, the RSD of relative peak area is between 0.20 and 2.95 percent, and the RSD is less than 5.0 percent. The above experimental results show that the reproducibility of the experiment according to the method of the invention is good.
5. Small knot
The fingerprint detection method of the Qigu capsule established by the invention meets the requirements on specificity, precision, stability and repeatability of the method, and the established method is feasible.
6. Establishing comparison fingerprint
(1) Establishing comparison fingerprint spectrum, and calibrating common peak
The test solution is prepared from 21 batches of Qigu capsules according to the test solution preparation method, and the results are shown in FIG. 5.
The measured chromatograms of 21 batches of Qigu capsules as test samples are introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (version 2012.130723) issued by the State pharmacopoeia Committee, the standard spectrum generation method is a median method, simultaneously, a multipoint correction method is adopted to establish a Qigu capsule contrast fingerprint, and the result is shown in figure 6.
According to the fingerprint analysis condition of the Qigu capsule, chromatographic peaks with good separation degree of main components in the Qigu capsule are selected as characteristic peaks, and 15 common peaks are determined. Comparing 15 main chromatographic peaks with each medicinal material and negative sample, identifying 14 peaks, wherein 7, 8, 9, 10, 11, 12, 13 and 14 belong to herba Epimedii; peak 4, 15 belongs to Polygonum multiflorum; peak 5, 6 belong to drynaria; peak 3 belongs to Astragalus membranaceus; peak 2 belongs to Cistanchis herba. In addition, a chemical composition of 11 peaks was identified, peak 2: echinacoside; peak 3: calycosin glucoside; peak 4: 2,3,5, 4-tetrahydroxystilbene glucoside; peak 6: naringin; peak 7: epimedin A; peak 8: epimedin B; peak 9: epimedin C; peak 10: icariin; peak 11: icariside I; peak 14: baohuoside I; peak 15: emodin is added.
2. Evaluation of similarity
The chromatograms of the 21 batches of Qigu capsules as test samples are introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (version 2012.130723) issued by the national pharmacopoeia committee, and the similarity between the chromatogram of each test sample and the reference chromatogram is calculated, and the result is shown in Table 12.
TABLE 12 evaluation results of similarity
Figure BDA0002616946840000141
Watch 12 (continuation)
Figure BDA0002616946840000151
The similarity between the test sample spectrum and the comparison fingerprint spectrum is respectively 0.997, 0.990, 0.994, 0.992, 0.990, 0.987, 0.991, 0.994, 0.995, 0.988, 0.980, 0.998, 0.996, 0.995, 0.997, 0.998, 0.991, 0.989 and 0.971, which are all more than 0.9, which indicates that the 21 batches of astragalus bone capsules have stable and uniform quality.

Claims (8)

1. A liquid chromatography method for detecting Qigu capsule fingerprint spectrum adopts a high performance liquid chromatograph equipped with an ultraviolet detector to carry out detection, and the chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX SB-C18 column, 250mm 4.6mm, 5 μm,
column temperature: 30 deg.C
Flow rate: 1 ml/min;
detection wavelength: 270 nm;
sample introduction amount: 10 μ l
Mobile phase: methanol (A) -acetonitrile (B) -0.1% phosphoric acid aqueous solution (C)
Gradient elution conditions:
t/min methanol% Acetonitrile% 0.1% phosphoric acid aqueous solution% 0 4 1 95 10 11 7 82 33 11 19 70 47 16 24 60 57 25 34 41 67 56 34 10 77 4 1 95
。
2. The liquid chromatography method for detecting the fingerprint of the Qigu capsule as claimed in claim 1, wherein the sample solution of the Qigu capsule for detection is prepared as follows: extracting the content of the astragalus-bone capsule with methanol according to a liquid-solid ratio of 50-200 times, wherein the liquid-solid ratio is a volume/mass ratio taking ml/g as a unit.
3. The liquid chromatography method for detecting the fingerprint of the Qigu capsule as claimed in claim 1, wherein the sample solution of the Qigu capsule is prepared as follows: weighing 0.5g of radix astragali bone capsule content, precisely weighing, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, supplementing loss mass with methanol, and filtering with 0.45 μm microporous membrane to obtain test solution.
4. A method of establishing a stilbene bone capsule control fingerprint, the method comprising:
(1) preparing a test solution:
weighing 0.5g of radix astragali bone capsule content, precisely weighing, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, supplementing loss mass with methanol, and filtering with 0.45 μm microporous membrane;
(2) preparing a reference substance solution:
respectively taking a proper amount of echinacoside, calycosin glucoside, 2,3,5, 4-tetrahydroxystilbene glucoside, naringin, epimedin A, epimedin B, epimedin C, icariin, icarisid I, baohuoside I and emodin, precisely weighing, and adding methanol to prepare a mixed solution containing 18.55 mu g of echinacoside, 25.22 mu g of calycosin glucoside, 30.95 mu g of 2,3,5, 4-tetrahydroxystilbene glucoside, 26.89 mu g of naringin, 25.75 mu g of epimedin A, 20.00 mu g of epimedin B, 22.48 mu g of epimedin C, 18.92 mu g of icariin, 26.60 mu g of icarisid I, 22.70 mu g of baohuoside I and 16.07 mu g of emodin in each 1 ml;
(3) preparation of negative sample solution:
taking 0.5g of negative sample powder lacking herba Epimedii, radix Polygoni Multiflori Preparata, radix astragali, herba Dendrobii, Cistanchis herba, rhizoma Drynariae or flos Chrysanthemi, precisely weighing, precisely adding 50ml of methanol, weighing, performing ultrasound for 30min, supplementing the loss mass with methanol, filtering with 0.45 μm microporous membrane, and preparing each negative sample solution;
(4) preparing a medicinal material solution:
respectively extracting herba Epimedii, radix Polygoni Multiflori Preparata, radix astragali, herba Dendrobii, Cistanchis herba, rhizoma Drynariae, and flos Chrysanthemi with water under reflux, concentrating, filtering with 0.45 μm microporous membrane, and making into solution;
(5) and (4) HPLC detection:
respectively and precisely absorbing 10 mu l of each of the test solution, the reference solution, the negative sample solution and the medicinal material solution of the (1), (2), (3) and (4), and detecting by using the liquid chromatography method for detecting the fingerprint of the stilbene bone capsule to obtain a test sample fingerprint chromatogram, a reference fingerprint chromatogram, a negative sample fingerprint chromatogram and a medicinal material fingerprint chromatogram;
(6) generating a standard fingerprint spectrum:
generating a comparison fingerprint spectrum based on common peaks in fingerprint chromatograms of N batches of test samples, wherein chromatographic peaks with good separation degree of main components in the Qigu capsules are selected as characteristic peaks, and 15 common peaks are determined, wherein N is more than 10, and is preferably 10-30;
(7) identification and attribution of common peaks:
and (4) attributing and identifying common peaks in the Qigu capsule fingerprint.
5. The method for establishing the Qigu capsule contrast fingerprint spectrum of claim 4, wherein the identification and attribution of the common peaks are completed by comparing with the chromatograms of the negative sample, the single medicinal material and the contrast solution.
6. A radix astragali and bone capsule control fingerprint spectrum is shown in figure 6, and comprises 15 characteristic peaks.
7. The Qigu capsule control fingerprint spectrum of claim 6, which is obtained by the method for establishing the Qigu capsule control fingerprint spectrum of claim 4.
8. A quality control method of Qigu capsules comprises the following steps:
(1) preparing a test solution:
taking 0.5g of the content of the stilbene bone capsule sample to be detected, precisely weighing, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, complementing loss mass with methanol, and filtering through a 0.45 mu m microporous membrane;
(2) chromatographic conditions are as follows:
adopting the chromatographic conditions in the liquid chromatographic method for detecting the fingerprint of the Qigu capsule according to claim 1;
(3) and (3) determination:
precisely sucking 10 mu l of the solution (1), injecting into a liquid chromatograph, and measuring to obtain a fingerprint of the stilbene bone capsule sample to be detected;
(4) and (4) qualification judgment:
and calculating the similarity between the fingerprint of the stilbene bone capsule sample to be detected and the reference fingerprint, wherein the similarity is more than or equal to 0.8, and obtaining a qualified product.
CN202010771890.1A 2020-08-04 2020-08-04 Radix astragali-bone capsule fingerprint detection method, control fingerprint and application Pending CN114062525A (en)

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