CN112067725B - Standard fingerprint spectrum construction method of Yitong tablet and method for measuring its component content - Google Patents
Standard fingerprint spectrum construction method of Yitong tablet and method for measuring its component content Download PDFInfo
- Publication number
- CN112067725B CN112067725B CN202010990800.8A CN202010990800A CN112067725B CN 112067725 B CN112067725 B CN 112067725B CN 202010990800 A CN202010990800 A CN 202010990800A CN 112067725 B CN112067725 B CN 112067725B
- Authority
- CN
- China
- Prior art keywords
- peak
- tablet
- yitong
- standard fingerprint
- methanol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a construction method of standard fingerprint of a yitong tablet and a method for measuring the component content of the yitong tablet, which comprises the following steps: preparing a test solution; preparation of control solutions: dissolving barbaloin in mixed solution of methanol and 0.1% volume concentration acetic acid water, respectively dissolving naringin, neohesperidin and echinacoside in methanol to obtain reference solutions; and (3) high performance liquid chromatography detection: the chromatographic conditions are as follows: adopting a mobile phase A as methanol and a mobile phase B as 0.1% volume concentration acetic acid aqueous solution, wherein the total flow rate is 1 ml/min; performing gradient elution; establishing standard fingerprint of the yitong tablet; the fingerprint spectrum of the invention has better peak type, better separation degree, symmetrical factor and theoretical plate number; the method can effectively separate the components with smaller structural difference, can better improve the quality of standard products, and can more accurately realize the simultaneous determination of the content of the effective components in the products.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a construction method of standard fingerprint spectrums of yitong tablets and a method for measuring component content of the yitong tablets.
Background
The constipation is a common disease and frequently encountered disease, and a medicine for effectively treating the constipation is lacked, so that the innovative traditional Chinese medicine mainly for treating the constipation is developed by relying on the theory of the traditional Chinese medicine, is oriented to market demands, and is an important pioneer for inheriting and developing the tradition of the traditional Chinese medicine. The innovative traditional Chinese medicine 'yitong tablet' is an innovative traditional Chinese medicine mainly used for treating constipation, and has already obtained clinical research batch, so that the research and development of the innovative traditional Chinese medicine have great strategic significance for the sustainable development of the traditional Chinese medicine industry. The yitong tablet comprises 3-6 parts by weight of aloe, 6-11 parts by weight of immature bitter orange, 6-11 parts by weight of cassia seed, 6-11 parts by weight of fructus cannabis, 6-11 parts by weight of cistanche, 6-11 parts by weight of peach kernel, 6-11 parts by weight of angelica sinensis, 18-25 parts by weight of radix scrophulariae and 18-25 parts by weight of radix rehmanniae recen.
The quality standard of the existing Chinese patent medicines mostly adopts the content of single index components, and does not meet the action characteristics of multiple components, multiple targets and multiple ways of the traditional Chinese medicine. The HPLC fingerprint spectrum of the traditional Chinese medicine is a comprehensive, integral and quantifiable quality evaluation method which adopts a high performance liquid chromatography method to calibrate the common characteristic peak spectrum of the traditional Chinese medicine, can comprehensively reflect the main component conditions of the Chinese patent medicine preparation. At present, Chinese patent medicine quality evaluation is carried out by utilizing the fingerprint of the Chinese medicine, which shows that the consistency among batches is widely accepted and accepted at home and abroad. The quality standard of the existing product only quantitatively measures ferulic acid in a prescription, has simple standard and low ferulic acid content, is difficult to comprehensively control the product quality, and has no quality evaluation standard conforming to the characteristics of the traditional Chinese medicine.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a construction method of a standard fingerprint of a tonable tablet and a method for measuring the component content of the tonable tablet.
The invention provides a standard fingerprint construction method of a yitong tablet, which comprises the following steps:
1) preparation of a test solution: taking the Yitong tablets, removing the coating, grinding, mixing with methanol, carrying out ultrasonic treatment, fixing the volume, filtering, and taking the filtrate to obtain a test solution;
2) preparation of control solutions: dissolving aloin in mixed solution of methanol and 0.1% volume concentration acetic acid water, respectively dissolving naringin, neohesperidin and echinacoside in methanol to obtain aloin, naringin, neohesperidin and echinacoside reference solution;
3) and (3) high performance liquid chromatography detection:
the chromatographic conditions are as follows: the chromatographic column is C18A reverse phase chromatography column; the column temperature was 25 ℃; the detection wavelength is 300nm, the mobile phase A is methanol, the mobile phase B is 0.1% acetic acid aqueous solution by volume concentration, and the total flow rate is 1 ml/min; gradient elution was performed to the extent shown in table 1 below:
TABLE 1 gradient elution schedule
4) Establishing a standard fingerprint spectrum of the Yitong tablets: sucking 10 μ L of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, detecting by the high performance liquid chromatography of the step 3), matching 16 common peaks, determining 6 characteristic peaks, and establishing standard fingerprint of the yitong tablet.
Preferably, in the method for preparing the test solution in step 1), the mass concentration of the test is 0.02 g/ml.
Preferably, in the method for preparing the test solution in step 1), the power of the ultrasonic treatment is 500w, the frequency is 40kHz, and the temperature of the ultrasonic treatment is lower than 25 ℃.
Preferably, in the chromatographic conditions of step 3), the chromatographic column is Cl8The column length of the chromatographic column was 25cm, the inner diameter was 4.6mm, and the particle size was 5 μm.
Preferably, in the step 2) of preparing the reference solution, the volume ratio of the mixed solution of methanol and 0.1% by volume of acetic acid is 39: 61.
Preferably, the mass concentration of the barbaloin is 0.04618-0.9235 mg/mL, the mass concentration of the naringin is 0.03780-0.7561 mg/mL, the mass concentration of the neohesperidin is 0.03755-0.7510 mg/mL, and the mass concentration of the echinacoside is 0.02598-0.5196 mg/mL.
Preferably, the standard fingerprint spectrum of the Yitongtian tablets has 6 characteristic peaks, wherein the peak 2 is echinacoside, the peak 4 is naringin, the peak 5 is neohesperidin, the peak 9 is barbaloin, the peak 15 is aloe-emodin, and the peak 16 is rhein;
TABLE 2 relative retention time of 6 characteristic peaks of the characteristic map
Taking peak number 9 as a reference peak, the relative retention time of 6 characteristic peaks of the accessible standard fingerprint is shown in table 2.
A method for determining the content of component of TONGTONG tablet by standard fingerprint comprises calculating the contents of echinacoside, naringin, neohesperidin and aloin according to standard fingerprint.
At present, in the construction of the fingerprint of traditional Chinese medicines, especially traditional Chinese medicine mixtures, because the traditional Chinese medicines comprise a plurality of raw materials, in order to control the product quality, the effective components are often difficult to determine, or the peak shapes, the separation degrees and the like of the plurality of effective components are not good, so that one component of ferulic acid is generally used as the quality standard at present, and the result is not ideal.
The invention has the beneficial effects that:
the invention aims at establishing the standard fingerprint spectrum of the yitong tablets and simultaneously measuring the content of 4 chemical components, deepens the quality standard, accurately controls the indexes, realizes the Chinese patent medicine yitong tablet quality evaluation system which accords with the characteristics of the traditional Chinese medicine, improves the product quality and stabilizes the quality.
The invention adopts multi-component analysis methods such as traditional Chinese medicine fingerprint spectrum technology and the like, and is based on the establishment method of the standard fingerprint spectrum of the yitong tablets; setting the content range of active components of Chinese medicine, and establishing a content measuring method for simultaneously measuring 4 chemical components. The HPLC fingerprints of different batches of the alunite tablets are researched for the first time, the alunite tablet fingerprints are established, main common peaks are assigned, and the barbaloin, naringin, neohesperidin and echinacoside which are main components are quantitatively measured, so that the quality standard is deepened, the indexes are accurately controlled, a Chinese patent medicine alunite tablet quality evaluation system which accords with the characteristics of the traditional Chinese medicine is realized, the product quality is improved, and the quality is stabilized.
According to the invention, 10 batches of fingerprints of the yitong tablets are established, 16 common peaks are calibrated by adopting fingerprint software, the similarity is more than 0.95, the established fingerprints have better controllability, and a basis is provided for quality evaluation of the yitong tablets. The methodology experiment shows that the chromatographic method can simultaneously quantitatively analyze the contents of barbaloin, naringin, neohesperidin and echinacoside. In conclusion, by adopting the HPLC fingerprint method established above, fingerprint detection and effective component content determination are simultaneously carried out on the yitong tablets, medicinal materials, decoction pieces and finished products, so that transition from terminal quality control to production process quality control is realized, product quality is improved, quality is stabilized, and a foundation is laid for marketing production of the yitong tablets.
The similarity of the fingerprint spectrum is very high, which shows that the selection of all common peaks is very good and the product commonality is high; the fingerprint spectrum of the invention has better peak type, better separation degree, symmetrical factor and theoretical plate number; the method can effectively separate the components with smaller structural difference, can better measure the quality of the standard product and more accurately measure the content of the effective components of the product. The method utilizes the HPLC fingerprint to control the quality of the Yitong tablets, establishes the standard map of the Yitong tablets, evaluates the similarity, simultaneously realizes the simultaneous determination of the content of 4 index components, more comprehensively reflects the quality of the Yitong tablets compared with the prior related reports, and ensures the consistency among the preparation batches.
Drawings
FIG. 1 shows HPLC finger prints of 10 lots of Yuantong tablets.
FIG. 2 is a comparison graph of standard fingerprint of TONGTONG tablet and a reference substance.
FIG. 3 is a comparison graph of standard fingerprint of TONGTONG tablet and original medicinal material.
FIG. 4 is an HPLC fingerprint of comparative example 1.
FIG. 5 is an HPLC fingerprint of comparative example 2.
FIG. 6 is an HPLC fingerprint of comparative example 3.
FIG. 7 is an HPLC fingerprint of comparative example 4.
FIG. 8 is a fingerprint of the aloin control solution at 0 h.
FIG. 9 is a fingerprint of 4h barbaloin control solution in methanol.
FIG. 10 is a fingerprint of 4h barbaloin control solution with mobile phase as solvent.
Detailed Description
The following examples are presented to further illustrate the embodiments of the present invention and are not intended to limit the invention thereto.
Example 1
Establishment of standard fingerprint spectrum of Yitong tablet
In order to make the medicinal materials have enough representativeness, medicinal materials of different producing areas or medicinal materials of different commodity specification grades are collected as test products.
Chromatographic conditions
A chromatographic column: InertSustain C18 (4.6X 250mm, 5 μm) mobile phase: methanol-0.1 acetic acid water gradient elution: 0-10 min: 20 to 40 percent of methanol; 10-20 min: 40% methanol; 20-30 min: 40 to 42 percent of methanol; 30-50 min: 42 to 47 percent of methanol; 50-80 min: 47% -60% of methanol; 80-90 min: 60 to 90 percent of methanol; and (5) 90-100 min: 90% methanol. Detection wavelength: 300 nm; flow rate: 1 ml/min; column temperature: 25 ℃; the sample amount is 10 mul; the theoretical plate number is not less than 5000 calculated by barbaloin.
Preparation of the solution
Preparation of control solutions
Precisely weighing appropriate amounts of aloin, naringin, neohesperidin, echinacoside, rhein and aloe-emodin reference substances, dissolving aloin with a mobile phase methanol-0.1% acetic acid water mixed solution (39:61) and fixing the volume to prepare a reference substance solution containing the aloin with the mass concentration of 0.9235mg/mL, dissolving the other reference substances with methanol and fixing the volume to prepare a reference substance solution containing the naringin with the mass concentration of 0.7561mg/mL, the neohesperidin with the mass concentration of 0.7510mg/mL, the echinacoside with the mass concentration of 0.5196mg/mL, the rhein with the mass concentration of 0.56mg/mL and the aloe-emodin with the mass concentration of 0.56 mg/mL.
Preparation of test solution
Taking 20 tablets of the product, removing the coating, grinding, taking about 1g, precisely weighing, placing in a 50ml brown measuring flask, adding a proper amount of solvent, carrying out ultrasonic treatment (power 500W, frequency 40kHz, temperature lower than 25 ℃) for 20 minutes, cooling, adding methanol to dilute to a scale mark, shaking up, filtering, and taking a subsequent filtrate to obtain the product.
Methodology investigation
Precision test
Precisely sucking 10 μ L of the same sample solution (S1), continuously feeding sample for 6 times according to chromatographic conditions, and recording chromatogram. The relative retention time and the relative peak area of 16 common peaks are calculated by taking the No. 9 peak (barbaloin) as a reference, and the result shows that the RSD of the relative retention time and the relative peak area of each characteristic peak are respectively 0.01-0.07% and 0.91-9.38%, wherein the RSD of the peak areas of the barbaloin, the naringin, the neohesperidin and the echinacoside are respectively 2.16%, 1.16%, 1.15% and 2.86%, and the instrument has good precision and meets the requirement of a fingerprint spectrum.
Repeatability test
Taking 6 parts of TONGTONG tablet (S1), precisely weighing, preparing sample solution, precisely sucking 10 μ L of sample solution, respectively sampling, measuring, and recording chromatogram. The relative retention time and the relative peak area of 16 common peaks are calculated by taking the 9 peak (barbaloin) as a reference, and the results show that the RSD of the relative retention time and the relative peak area of each characteristic peak is 0.01-0.14% and 1.16-9.81% respectively. The RSD of the peak areas of the barbaloin, the naringin, the neohesperidin and the echinacoside are respectively 0.88%, 1.83%, 2.10% and 1.50%, and the result shows that the method has good repeatability.
Stability test
Precisely sucking 10 μ L of the same sample solution (S1) for testing at 0, 4, 8, 12, 16, and 24h, and recording chromatogram. The relative retention time and the relative peak area of 16 common peaks are calculated by taking the peak No. 9 (barbaloin) as a reference, and the results show that the RSD of the relative retention time and the relative peak area of each characteristic peak is 0.02-0.27% and 1.49-8.14% respectively. The RSD of the peak areas of the barbaloin, the naringin, the neohesperidin and the echinacoside are respectively 3.87%, 2.50%, 2.40% and 3.97%, and the result shows that the test result of the test solution is stable within 24 h.
Determination of finger print
Establishment of standard fingerprint of TONGTIAN tablet 10 batches of TONGTIAN tablet samples (S1-S10) are precisely weighed, sample solution is prepared, 10 μ L of sample solution is precisely absorbed, the detection wavelength is 300nm, and the sample is respectively injected and measured, and the spectrum of 100min is recorded. And (3) performing data analysis processing by using a Chinese medicine chromatography fingerprint similarity evaluation system (2004A) of the national pharmacopoeia committee, setting S1 as a reference spectrum, and automatically matching the chromatographic peaks of other samples with the reference spectrum to generate a cartoon fingerprint, wherein a peak 9 is taken as a reference peak (S) as shown in figure 1.
The relative retention time and relative peak area of each common peak were measured as shown in tables 1 and 2.
TABLE 110 batch size Yitong tablets HPLC fingerprint chromatogram common peak relative retention time
TABLE 210 batch KOUITONG tablet HPLC fingerprinting common peak relative peak area
Determination of common peaks 16 common peaks are marked according to the fingerprint detection results of 10 batches of the yitong tablets through similarity evaluation software, and the sample peaks are compared and analyzed with a reference substance, and the sample peaks indicate that the peak 2 is echinacoside, the peak 4 is naringin, the peak 5 is neohesperidin, the peak 9 is barbaloin, the peak 15 is aloe-emodin, and the peak 16 is rhein, which is shown in figure 2. Since peak 9 has a high degree of separation and a stable peak area, this peak was selected as a reference peak.
Evaluation of similarity of fingerprints the similarity of the S1-S10 samples and the fingerprints is calculated through software, the similarity between 10 groups of samples and the fingerprints is 0.971-0.999, and the similarity is good.
Example 2 control solvent stability study
Barbaloin reference solution (i): aloin control 1.23mg (purity 94.4%) → 10ml (methanol)
Barbaloin reference solution 2: aloin control 1.23mg (purity 94.4%) → 10ml (mobile phase)
And taking the reference substance solution, respectively carrying out sample injection analysis at 0h, 2h, 4h and 6h, and comparing peak areas to calculate the barbaloin degradation rate. The result shows that the barbaloin is more stable in the mobile phase mixed solution than in the methanol solution, and the literature shows that the barbaloin solution is sensitive to light and heat, so the mobile phase is required to be used as a solvent when preparing the barbaloin reference solution, and the barbaloin reference solution is required to be stored away from light and at low temperature.
TABLE 3 control solution stability study
The barbaloin is mostly the sum of two optical isomers, barbaloin A and barbaloin B, and barbaloin A has stronger biological activity than barbaloin B. In the experiment, methanol and a mobile phase methanol-0.1% acetic acid aqueous solution (39:61) are respectively used as solvents to dissolve the barbaloin reference substance, and the degradation rates of barbaloin in 0h, 2h, 4h and 6h are examined, and the results show that barbaloin is more stable in a mobile phase methanol-0.1% acetic acid aqueous solution (39:61) mixed solution than in the methanol solution, and the barbaloin reference substance solution is sensitive to light and heat, so the mobile phase is required to be used as the solvent when the barbaloin reference substance solution is prepared, and the barbaloin reference substance needs to be protected from light and is used as the current preparation.
Example 3
And confirming the sources of the common peaks, respectively taking a proper amount of 9 medicinal materials in the universal formula, precisely weighing, and preparing a medicinal material sample solution. Precisely sucking respectively, injecting 10 μ L of prepared sample solution and 9 medicinal material sample solution respectively, injecting into high performance liquid chromatograph, and recording chromatogram for 100 min. Analyzing and comparing chromatographic peaks with consistent retention time, identifying related peaks of the whole formula and the fingerprint of the medicinal material according to ultraviolet spectrum information, and confirming the attribution of medicinal materials with common peaks, as shown in figure 3.
Through the research on the correlation of the Yitongtian compound and the whole formula medicinal materials, the sources of 16 characteristic peaks in the HPLC fingerprint spectrum of the Yitongtian compound are confirmed, and the fingerprint peaks are respectively from cistanche deserticola (peak 2, peak 3), radix rehmanniae (peak 3), radix scrophulariae (peak 3), fructus aurantii immaturus (peak 4, peak 5, peak 7), aloe (peak 1, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16) and angelica (peak 16).
Example 4
Determination of sample content
Inspecting linear relationship, adding mobile phase (or methanol) into the solution to dilute into control solution with series mass concentration, measuring, injecting 10 μ L, measuring peak area integral value (Y), drawing standard curve with mass concentration as abscissa (X) and Y as ordinate, and performing linear regression to obtain regression equation of aloin Y-0.2007X +0.0187, wherein r is20.9999, which shows that the barbaloin has good linear relation at 0.4618-9.2354 mug; naringin y 0.1513x +0.3505, r 21, the naringin has a good linear relation between 0.3780 and 7.5607 mu g; neohesperidin y-0.1501 x +1.3668, r20.9999, which shows that the neohesperidin has a good linear relation between 0.3755 and 7.5070 mu g; echinacoside y-0.1866 x-0.2159, r2The result shows that echinacoside has a good linear relation between 0.2598 and 5.1959 mu g when the echinacoside is 0.9995.
Sample adding recovery rate test 0.5g and 6 parts of samples (S1) with known contents of aloin, naringin, neohesperidin and echinacoside are precisely weighed, 8.1mg of an aloin reference substance, 7.9mg of the naringin reference substance, 8.6mg of the neohesperidin reference substance and 3.5mL (0.5196mg/mL) of an echinacoside reference substance solution (namely 1.8 mg) are precisely added respectively, the mixture is placed in a 50mL brown measuring flask, a proper amount of solvent is added, ultrasonic treatment (with the power of 500W, the frequency of 40kHz and the temperature of lower than 25 ℃) is carried out for 20 minutes, the mixture is cooled, methanol is added for dilution to a scale line, the mixture is shaken up and filtered, and a subsequent filtrate is taken, thus obtaining the medicine. According to the analysis of chromatographic conditions, the average sample recovery rates of the barbaloin, the naringin, the neohesperidin and the echinacoside are respectively 101.62%, 99.52%, 102.05% and 98.35%, and the RSDs are respectively 2.01%, 1.95%, 2.21 and 2.84%, which shows that the method has better sample recovery rates of the four.
Sample content determination A proper amount of 10 batches of the alundum tablets were taken, precisely weighed, and a test solution was prepared and analyzed according to chromatographic conditions, and the results are shown in Table 4.
TABLE 4 results of content measurement (%)
Example 5
Selection of extraction conditions
According to the extraction solvent of the compound process and the preparation method of the test sample for measuring the barbaloin content in the revised quality standard draft, the research researches investigate different extraction modes (ultrasonic extraction and heating reflux extraction) of the yitong tablets and different extraction solvents (ethanol, water, methanol and 50% methanol), and the results show that the methanol ultrasonic extraction effect is optimal.
Selection of chromatographic conditions
By adopting a DAD full-wavelength scanning mode and referring to the detection wavelength of the effective components of each single traditional Chinese medicinal material in the prescription, spectra under the wavelengths of 200 nm, 220 nm, 235 nm, 260 nm, 280 nm, 300nm and the like are extracted and compared, and under 300nm, the chromatogram of a sample has a large number of chromatographic peaks and a high peak response value. The response values of echinacoside, naringin, neohesperidin and aloin at different wavelengths are shown in table 4.
TABLE 4 response values of echinacoside, naringin, neohesperidin, barbaloin at different wavelengths
4 mobile phase systems of methanol-water, acetonitrile-water, methanol-0.1% acetic acid water and methanol-0.2% acetic acid water are investigated, and the mobile phase system adopting the methanol-0.1% acetic acid water is found to have more chromatographic peaks, better reference peak (barbaloin) peak type, better separation degree, symmetry factor and theoretical plate number.
Identification of the source of the common peaks
The invention identifies the related peaks of the whole formula and the fingerprint of the medicinal material, and confirms the material components of 6 peaks, mainly from cistanche, immature bitter orange and aloe. The No. 8 and No. 9 peaks in the sample are isomers of barbaloin, barbaloin solution is sensitive to light and heat, and the barbaloin reference solution is prepared by using a mobile phase as a solvent, keeping out of the sun and storing at low temperature.
Comparative example
To illustrate the effectiveness of the elution procedure of the present application, different comparative examples 1-4 were tested, the elution procedures are shown in tables 5-8, respectively, and the other steps are the same as in example 1.
Table 5 elution schedule for comparative example 1
Table 6 elution schedule for comparative example 2
Table 7 elution schedule for comparative example 3
Table 8 elution schedule for comparative example 4
The fingerprints of the comparative examples 1 to 4 are respectively shown in fig. 4 to 7, and as can be seen from the differences between the fingerprints of the present application and the fingerprints of the present application, the fingerprint of the present application has a good peak pattern and a good degree of separation; the method can effectively separate the components with smaller structural difference, can better measure the quality of the standard product and more accurately measure the content of the effective components of the product.
Test examples
To illustrate the effect of different solvents on the stability of barbaloin, the present application performed comparative experiments with methanol as the solvent. The results of the tests are shown in comparison in FIGS. 8-10.
As can be seen from a comparison of fig. 9-10, the stability of barbaloin is better when the mobile phase of the present application is used as a solvent.
Claims (8)
1. A construction method of standard fingerprint of yitong tablets is characterized by comprising the following steps:
1) preparation of a test solution: taking the Yitong tablets, removing the coating, grinding, mixing with methanol, carrying out ultrasonic treatment, fixing the volume, filtering, and taking the filtrate to obtain a test solution;
the yitong tablet comprises 3-6 parts by weight of aloe, 6-11 parts by weight of immature bitter orange, 6-11 parts by weight of cassia seed, 6-11 parts by weight of fructus cannabis, 6-11 parts by weight of cistanche, 6-11 parts by weight of peach kernel, 6-11 parts by weight of angelica sinensis, 18-25 parts by weight of radix scrophulariae and 18-25 parts by weight of radix rehmanniae recen;
2) preparation of control solutions: dissolving aloin in mixed solution of methanol and 0.1% volume concentration acetic acid water, respectively dissolving naringin, neohesperidin and echinacoside in methanol to obtain aloin, naringin, neohesperidin and echinacoside reference solution;
3) and (3) high performance liquid chromatography detection:
the chromatographic conditions are as follows: the chromatographic column is C18A reverse phase chromatography column; the column temperature was 25 ℃; the detection wavelength is 300nm, the mobile phase A is methanol, the mobile phase B is 0.1% acetic acid aqueous solution by volume concentration, and the total flow rate is 1 ml/min; gradient elution was performed to the extent shown in table 1 below:
TABLE 1 gradient elution schedule
4) Establishing a standard fingerprint spectrum of the Yitong tablets: sucking 10 μ L of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, detecting by the high performance liquid chromatography of the step 3), matching 16 common peaks, determining 6 characteristic peaks, and establishing standard fingerprint of the yitong tablet.
2. The method for constructing standard fingerprint of TONGTONG tablet according to claim 1, wherein in the step 1), the sample solution is prepared at a concentration of 0.02 g/ml.
3. The method for constructing standard fingerprint of accessible slices as claimed in claim 1 or 2, wherein the preparation method of the test solution in step 1) comprises the steps of performing ultrasonic treatment at a power of 500w and a frequency of 40kHz, and performing ultrasonic treatment at a temperature of less than 25 ℃.
4. The method for constructing standard fingerprint of accessible slice according to claim 1 or 2, wherein in the chromatographic conditions of step 3), the chromatographic column is Cl8The column length of the chromatographic column was 25cm, the inner diameter was 4.6mm, and the particle size was 5 μm.
5. The method for constructing standard fingerprint of a tonable tablet according to claim 1 or 2, wherein the volume ratio of the mixed solution of methanol and 0.1% by volume of acetic acid water in the step 2) is 39: 61.
6. The method for constructing standard fingerprint of yitong tablets as claimed in claim 1 or 2, wherein the mass concentration of the aloin is 0.04618-0.9235 mg/mL, the mass concentration of the naringin is 0.03780-0.7561 mg/mL, the mass concentration of the neohesperidin is 0.03755-0.7510 mg/mL, and the mass concentration of the echinacoside is 0.02598-0.5196 mg/mL.
7. The method for constructing standard fingerprint of TONGTONG tablet according to claim 1 or 2, wherein the standard fingerprint of TONGTONG tablet has 6 characteristic peaks, wherein peak 2 is echinacoside, peak 4 is naringin, peak 5 is neohesperidin, peak 9 is barbaloin, peak 15 is aloe-emodin, and peak 16 is rhein;
TABLE 2 relative retention time of 6 characteristic peaks of the characteristic map
Taking peak number 9 as a reference peak, the relative retention time of 6 characteristic peaks of the accessible standard fingerprint is shown in table 2.
8. A method for measuring the content of the ingredients of the Tongbai tablet by using the standard fingerprint of the Tongbai tablet obtained by the construction method of any one of claims 1 to 7, which is characterized in that the contents of echinacoside, naringin, neohesperidin and barbaloin are calculated according to the standard fingerprint of the Tongbai tablet.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010990800.8A CN112067725B (en) | 2020-09-19 | 2020-09-19 | Standard fingerprint spectrum construction method of Yitong tablet and method for measuring its component content |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010990800.8A CN112067725B (en) | 2020-09-19 | 2020-09-19 | Standard fingerprint spectrum construction method of Yitong tablet and method for measuring its component content |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112067725A CN112067725A (en) | 2020-12-11 |
| CN112067725B true CN112067725B (en) | 2022-04-08 |
Family
ID=73681780
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010990800.8A Active CN112067725B (en) | 2020-09-19 | 2020-09-19 | Standard fingerprint spectrum construction method of Yitong tablet and method for measuring its component content |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112067725B (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1939461A (en) * | 2005-09-30 | 2007-04-04 | 北京奇源益德药物研究所 | Quality control of Chinese medicinal compound preparation |
| CN101979388A (en) * | 2010-06-28 | 2011-02-23 | 南京泽朗农业发展有限公司 | Method for purifying barbaloin |
| CN103965149A (en) * | 2014-04-30 | 2014-08-06 | 桂林军供生化技术开发有限公司 | Aloin extraction method |
| CN104181238A (en) * | 2013-05-22 | 2014-12-03 | 河北万邦复临药业有限公司 | Barbaloin detection method |
| CN108459090A (en) * | 2017-02-17 | 2018-08-28 | 华润三九医药股份有限公司 | The method of quality control of jichuan decoction composition |
| CN109254096A (en) * | 2018-10-25 | 2019-01-22 | 瑞阳制药有限公司 | The method of quality control of jichuan decoction preparation |
| CN109270181A (en) * | 2018-11-23 | 2019-01-25 | 鲁南制药集团股份有限公司 | The method for building up and its standard diagram of first luxuriant growth Tongbian capsule HPLC finger-print |
| CN109613166A (en) * | 2019-01-16 | 2019-04-12 | 鲁南制药集团股份有限公司 | A kind of head luxuriant growth Tongbian capsule quality determining method |
| CN110927274A (en) * | 2019-12-03 | 2020-03-27 | 健民药业集团股份有限公司 | Method for simultaneously detecting contents of six ingredients in shitong capsule |
| CN111624295A (en) * | 2020-07-02 | 2020-09-04 | 鲁南厚普制药有限公司 | Quality detection method of 'Jihui Tongbiang' capsule |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7776915B2 (en) * | 2005-03-24 | 2010-08-17 | Tracie Martyn International, Llc | Topical formulations and methods of use |
-
2020
- 2020-09-19 CN CN202010990800.8A patent/CN112067725B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1939461A (en) * | 2005-09-30 | 2007-04-04 | 北京奇源益德药物研究所 | Quality control of Chinese medicinal compound preparation |
| CN101979388A (en) * | 2010-06-28 | 2011-02-23 | 南京泽朗农业发展有限公司 | Method for purifying barbaloin |
| CN104181238A (en) * | 2013-05-22 | 2014-12-03 | 河北万邦复临药业有限公司 | Barbaloin detection method |
| CN103965149A (en) * | 2014-04-30 | 2014-08-06 | 桂林军供生化技术开发有限公司 | Aloin extraction method |
| CN108459090A (en) * | 2017-02-17 | 2018-08-28 | 华润三九医药股份有限公司 | The method of quality control of jichuan decoction composition |
| CN109254096A (en) * | 2018-10-25 | 2019-01-22 | 瑞阳制药有限公司 | The method of quality control of jichuan decoction preparation |
| CN109270181A (en) * | 2018-11-23 | 2019-01-25 | 鲁南制药集团股份有限公司 | The method for building up and its standard diagram of first luxuriant growth Tongbian capsule HPLC finger-print |
| CN109613166A (en) * | 2019-01-16 | 2019-04-12 | 鲁南制药集团股份有限公司 | A kind of head luxuriant growth Tongbian capsule quality determining method |
| CN110927274A (en) * | 2019-12-03 | 2020-03-27 | 健民药业集团股份有限公司 | Method for simultaneously detecting contents of six ingredients in shitong capsule |
| CN111624295A (en) * | 2020-07-02 | 2020-09-04 | 鲁南厚普制药有限公司 | Quality detection method of 'Jihui Tongbiang' capsule |
Non-Patent Citations (2)
| Title |
|---|
| Application of Chromatography Technology in the Separation of Active Components from Nature Derived Drugs;H.-Y. Zhao et al.;《Mini-Reviews in Medicinal Chemistry》;20101231;第10卷;第1223-1234页 * |
| HPLC-MS/MS法同时测定首荟通便胶囊中8种成份分析;彭艳 等;《世界中医药》;20200731;第15卷(第14期);第2048-2052页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112067725A (en) | 2020-12-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107149623B (en) | Content determination method of traditional Chinese medicine composition | |
| CN105699500B (en) | Method for measuring content of 7 components in wrinkled gianthyssop vital energy dropping pills by ultra-high performance liquid chromatography | |
| CN104950052B (en) | A kind of method that Dementholized mint oil dripping pill quality is detected with gas chromatograph | |
| CN108152399B (en) | Construction and detection method of UPLC (ultra performance liquid chromatography) characteristic spectrum of semen boitae medicinal material | |
| CN105675739A (en) | Construction method of HPLC specific chromatogram of traditional Chinese medicines treating wind-heat cold | |
| CN110927311B (en) | Construction method of UPLC (ultra performance liquid chromatography) characteristic spectrum of dogbane leaf medicinal material and method for measuring content of flavonoid component of dogbane leaf medicinal material | |
| CN113686989B (en) | Method for constructing feature map of Weixuening granule | |
| CN111855867B (en) | Method for establishing characteristic spectrum of traditional Chinese medicine or traditional Chinese medicine composition preparation and application | |
| CN112666277A (en) | Method for constructing and detecting HPLC (high performance liquid chromatography) characteristic spectrum of rhizoma cyperi medicinal materials, decoction pieces, standard decoction and formula granules | |
| CN112067725B (en) | Standard fingerprint spectrum construction method of Yitong tablet and method for measuring its component content | |
| CN108663440A (en) | Callicarpa nudiflora medicinal material UPLC fingerprint map constructions method and standard finger-print | |
| CN115524424A (en) | Capsella bursa-pastoris sample quality control method | |
| CN105158397A (en) | Method for evaluating quality of four raw medicinal materials of blood replenishing type Chinese herbal medicine compound preparation simultaneously | |
| CN110031577B (en) | Quality detection method and identification application of traditional Chinese medicine or traditional Chinese medicine composition preparation | |
| CN112415104B (en) | Characteristic spectrum, construction method and detection method of pyrrosia pedunculata medicinal material, decoction pieces, standard decoction and formula granules thereof | |
| CN109781884B (en) | Establishing method of Qianliexin capsule fingerprint and fingerprint thereof | |
| CN113341007A (en) | Method for measuring contents of multiple components in whole Chinese date seed nerve-soothing capsule based on HPLC (high performance liquid chromatography) characteristic spectrum | |
| CN110308213B (en) | Method for constructing fingerprint spectrum of kidney-nourishing and fetus-growing pill and application of fingerprint spectrum in quality detection | |
| CN111965269A (en) | Method for measuring oleanolic acid content and ursolic acid content in patrinia heterophylla by using ultra-high performance liquid chromatography | |
| CN107976494B (en) | Construction of standard characteristic spectrum of Kangfu tincture and quality detection method thereof | |
| CN110470751A (en) | Method that is a kind of while detecting 7 kinds of component contents in sharp capsule for treating gastropathy | |
| CN109900832A (en) | A kind of detection method of rhizoma pinellinae praeparata finger-print | |
| CN115508471B (en) | Method for establishing characteristic spectrum of volatile components of blood activating and pain relieving tincture and application thereof | |
| CN113917012B (en) | Characteristic spectrum of Dingkundan, construction method and application thereof | |
| CN109725100B (en) | Method for detecting fingerprint spectrum of hawthorn granules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |