CN111487351A - Method for detecting fingerprint of blood-activating pain-relieving capsule - Google Patents
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Abstract
The invention discloses a method for detecting a fingerprint of a blood-activating pain-relieving capsule, which comprises the following steps: step 1, preparing a blood-activating pain-relieving capsule test solution; step 2, preparation of a single-standard reference substance solution: step 3, precisely absorbing the test solution to be injected into a liquid chromatograph, and recording a chromatogram; step 4, leading out the fingerprint instrument of the blood-activating pain-relieving capsule obtained in the step 3, leading the fingerprint instrument into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and selecting chromatographic peaks existing in chromatograms of different batches of blood-activating pain-relieving capsules as common peaks; generating a control fingerprint of the blood-activating pain-relieving capsule by using an average calculation method; the relative retention time and the relative peak area of each common peak were calculated. The fingerprint spectrum of the blood-activating pain-relieving capsule provided by the invention can comprehensively and objectively represent the quality of the blood-activating pain-relieving capsule. The fingerprint detection method provided by the invention has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like.
Description
Technical Field
The invention relates to a detection method of a traditional Chinese medicine preparation, in particular to a detection method of a finger print of a blood circulation promoting and pain relieving capsule.
Background
The fingerprint refers to a chromatogram or a spectrogram which can mark the chemical characteristics of certain complex substances, such as traditional Chinese medicines, DNA of certain organisms or certain tissues or cells, and proteins after being properly processed and by adopting a certain analysis means. The traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification means, is established on the basis of the systematic research of the chemical components of the traditional Chinese medicine, and is mainly used for evaluating the authenticity, the excellence and the stability of the quality of the traditional Chinese medicine and the traditional Chinese medicine preparation. The traditional Chinese medicine and the preparation thereof are all multi-component complex systems, so that the quality of the traditional Chinese medicine and the preparation thereof is evaluated by adopting a detection method which is adaptive to the traditional Chinese medicine and can provide rich identification information, and the establishment of the traditional Chinese medicine fingerprint spectrum can comprehensively reflect the types and the quantities of chemical components contained in the traditional Chinese medicine and the preparation thereof, thereby integrally describing and evaluating the quality of the medicine.
The prescription of the blood-activating and pain-relieving capsule (the national standard of medicine is Z10920002) is firstly seen in Zhao Xuannan clinical experience Collection, is a classic prescription and a first-choice medicine for treating traumatic injury and blood stasis and gall in traditional Chinese medicine, and is evaluated as a leading product in China-a user-preferred brand. The research mainly aims at the fingerprint spectrum of the main components of the product to better control the product quality.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the defects of the prior art and provides a fingerprint detection method of a blood-activating pain-relieving capsule, which can objectively, comprehensively and accurately evaluate the quality of the blood-activating pain-relieving capsule and has important significance for controlling the quality of the blood-activating pain-relieving capsule and ensuring the clinical curative effect.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:
A detection method of a fingerprint of a blood circulation promoting and pain relieving capsule is characterized by comprising the following steps:
Step 1, preparation of a blood-activating pain-relieving capsule test solution:
taking contents of the blood-activating and pain-relieving capsules of different batches, precisely weighing 0.5g of a content sample of the blood-activating and pain-relieving capsules respectively, placing the sample in a 100m L conical flask, adding 500m L of methanol, carrying out ultrasonic treatment for 60min, standing, taking supernate, and filtering the supernate with a 0.22 mu m microporous filter membrane to obtain a sample solution;
Step 2, preparation of a single-standard reference substance solution:
precisely weighing α -pinene, borneol, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 reference substances, placing in a volumetric flask, adding methanol to constant volume to scale, shaking up, and making into single-standard reference substance solution;
Step 3, precisely absorbing the test solution and the reference solution respectively, injecting the test solution and the reference solution into a high performance liquid chromatograph, and recording a chromatogram;
Step 4, exporting the fingerprint of the blood-activating pain-relieving capsule test solution obtained in the step 3, and introducing the fingerprint into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of blood circulation promoting and pain relieving capsules as common peaks; generating a control fingerprint of the blood-activating and pain-relieving capsule by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the single-standard comparison product solution chromatogram.
preferably, the method for detecting fingerprint of blood circulation promoting and pain relieving capsule in step 1 comprises the steps of taking 18 batches of blood circulation promoting and pain relieving capsules, precisely weighing 0.5g of a content sample of the blood circulation promoting and pain relieving capsule, placing the sample in a 100m L conical flask, adding methanol 500m L, carrying out ultrasonic treatment for 60min, standing, taking supernatant, and filtering with a 0.22 mu m microporous filter membrane to obtain a sample solution.
preferably, the method for detecting fingerprint of blood-activating pain-relieving capsule comprises the step 2 of preparing a single-standard reference solution, namely, preparing a precisely-weighed α -pinine, borneol, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1, putting the reference solution into a volumetric flask, fixing the volume to scale by using methanol, shaking up uniformly, and preparing the single-standard reference solution of 1.1296mg/m L alpha-pinine, 0.4136mg/m L borneol, 0.4092mg/m L notoginsenoside R1, 0.3588mg/m L ginsenoside Re, 0.4160mg/m L ginsenoside Rb1 and 0.4164mg/m L ginsenoside Rg 1.
As A preferred scheme, in the step 3, the liquid chromatography conditions of the method for detecting the fingerprint of the blood-activating and pain-relieving capsule are that A chromatographic column is YMC-PackODS-A, A mobile phase is acetonitrile (A) and 0.1% phosphoric acid water (B), an ultraviolet detector is used, the detection wavelength is 203nm, the column temperature is 35 ℃, the flow rate is 1.0m L/min, the sample injection volume is 10 mu L, and the gradient elution program is as follows:
| Procedure for measuring the movement of a moving object | Time (min) | Acetonitrile concentration (%) |
| 1 | 0.01 | 15 |
| 2 | 5.00 | 15 |
| 3 | 40.00 | 40 |
| 4 | 90.00 | 80 |
| 5 | 110.00 | 100 |
| 6 | 120.00 | 100 |
。
Preferably, the method for detecting fingerprint of the blood-activating pain-relieving capsule is characterized in that the fingerprint contains 17 peaks.
preferably, the method for detecting fingerprint of blood-activating pain-relieving capsule includes that the retention time of ginsenoside Rg1 is 21.35min and is peak 1, the retention time of notoginsenoside R1 is 25.62min and is peak 3, the retention time of ginsenoside Re is 27.46min and is peak 4, the retention time of ginsenoside Rb1 is 39.83min and is peak 5, the retention time of borneol is 79.74min and is peak 11, and the retention time of α -pinin is 92.40min and is peak 12.
Optimizing fingerprint detection conditions:
1. In the aspect of preparation optimization of sample solution
According to the invention, through experimental comparison of different extraction methods (ultrasonic extraction, reflux extraction, percolation and the like) and different extraction solvents (methanol, water, 70% ethanol aqueous solution, 85% ethanol aqueous solution, 95% ethanol, absolute ethanol), the results show that the spectrogram difference obtained by ultrasonic extraction and reflux extraction is small, and the ultrasonic extraction efficiency is high, so that the ultrasonic extraction method is adopted; the investigation of the extraction solvent finds that the chromatogram map of the methanol extract has the most information content and the highest component content; therefore, methanol is selected for extraction.
2. In the aspect of optimizing chromatographic conditions
According to the invention, a diode array detector is adopted to inspect the detection wavelength, chromatogram maps at positions of 203nm and 254nm are extracted, and when the detection wavelength is 203nm, the information content contained in the chromatogram maps is the most comprehensive, the detected components are the most, so 203nm is selected as the detection wavelength;
the flow rates (1m L/min, 0.8m L/min, 0.7m L/min, 0.6m L/min and 0.5m L/min) are screened, and because the components in the blood circulation-promoting and pain-relieving capsule mostly contain isomers and other components with extremely similar polarities, the components cannot be separated at a high flow rate, so that the separation effect is better at a low flow rate, and finally, the substances with similar polarities are separated under gradient conditions of the flow rate of 1m L/min for multiple times and the like.
The invention compares the elution effects of 5 different elution systems of methanol-water, acetonitrile-0.1% formic acid, acetonitrile and 0.05% phosphoric acid water, and acetonitrile-0.1% phosphoric acid water under different gradients. As a result, the acetonitrile and 0.1% phosphoric acid water are used as the mobile phase, the components in the blood-activating and pain-relieving capsule can be well separated, so that the acetonitrile and the water are finally selected as the mobile phase.
On the premise of optimizing the optimal mobile phase, the optimal gradient elution program is screened out through a large amount of experiments, and the volume concentration of acetonitrile is 15% when the time is 0.01-5 min; when the time is 5-40 min, the concentration of the acetonitrile is 15-40%; when the time is 40-90 min, the concentration of the acetonitrile is 40-80%; when the time is 90-110 min, the concentration of the acetonitrile is 80-100%; when the concentration of the acetonitrile is 100-100% in 110-120 min, the obtained fingerprint spectrum peak is the most complete, and the separation degree is the best.
Has the advantages that:
1. According to the structural property characteristics of active ingredients contained in the blood-activating pain-relieving capsule, the optimal mobile phase composition is screened out through a large number of experiments, and analysis conditions such as gradient elution procedures, flow rate, detection wavelength, chromatographic column, column temperature and the like are verified through a plurality of experiments.
2. The fingerprint of blood-activating pain-relieving capsule established by the method provided by the invention can effectively represent the quality of the blood-activating pain-relieving capsule, objectively reflect the front and back sequence and the mutual relation of each characteristic peak of the formed fingerprint, pay attention to the overall facial features, avoid the one-sidedness of judging the quality of the blood-activating pain-relieving capsule due to the measurement of individual chemical components, and reduce the possibility of manual treatment for reaching the quality standard.
3. The method for detecting the fingerprint of the blood-activating pain-relieving capsule provided by the invention has the advantages of simple method, good stability, high precision, good reproducibility and the like.
Drawings
FIG. 1 is a comparison fingerprint of a sample of the blood-activating pain-relieving capsule of the present invention.
FIG. 2 shows the fingerprint of 18 test samples of the blood-activating pain-relieving capsule of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not specified, according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The instruments and reagents used in the examples were as follows:
Experimental equipment
1.1 instruments
A dual-wavelength scanning high-performance liquid chromatography system of Shimadzu corporation in Japan comprises a full-automatic online degassing system, a full-automatic sample injection system SI L-20A, an ultraviolet detector SPD-20A, an automatic temperature control column incubator CTD-20AC, KH-500E type ultrasonic cleaner (Kunshan Seama ultrasonic instruments Co., Ltd.), and an M L104/02 electronic analytical balance (Mettler Toledo).
1.2 drugs and reagents
the blood-activating pain-relieving capsule sample sources are shown in Table 1, α -pinene (batch No. 110897-200502) is purchased from China institute for testing and testing food and medicine, borneol (batch No. 110881-201709) is purchased from China institute for testing and testing food and medicine, notoginsenoside R1 (batch No. 110745-201123) is purchased from China institute for testing and testing biological products of medicine, ginsenoside Re (batch No. 110754-201123) is purchased from China institute for testing and testing food and medicine, ginsenoside Rb1 (batch No. 110704-200424) is purchased from China institute for testing biological products of medicine, ginsenoside Rg1 (batch No. 110754-201123) is purchased from China institute for testing biological products of medicine, methanol (analytical purity), acetonitrile (chromatographic purity), and water (Wahaha purified water).
TABLE 1 blood circulation promoting and pain relieving Capsule sample information Table
Embodiment 1 a method for detecting fingerprint of blood circulation promoting and pain relieving capsule, comprising the following steps:
Step 1, preparation of a blood-activating pain-relieving capsule test solution:
taking 18 batches of the blood-activating and pain-relieving capsules in the table 1, precisely weighing 0.5g of the content sample of the 18 batches of the blood-activating and pain-relieving capsules respectively, placing the sample in a 100m L conical flask, adding methanol 500m L, carrying out ultrasonic treatment for 60min, standing, taking supernatant, and filtering the supernatant through a 0.22 mu m microporous filter membrane to obtain a sample solution.
Step 2, preparation of a single-standard reference substance solution:
precisely weighed α -pinine, borneol, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 reference substances, placing the reference substances in a volumetric flask, fixing the volume to scale by using methanol, and shaking up uniformly to prepare 1.1296mg/m L single-standard reference substance solutions of alpha-pinine, 0.4136mg/m L borneol, 0.4092mg/m L notoginsenoside R1, 0.3588mg/m L ginsenoside Re, 0.4160mg/m L ginsenoside Rb1 and 0.4164mg/m L ginsenoside Rg 1.
and 3, precisely sucking 15 batches of blood circulation promoting and pain relieving capsule test solution and reference solution respectively, injecting the test solution and the reference solution into A high performance liquid chromatograph, and recording A chromatogram, wherein the conditions of the liquid chromatogram comprise A chromatographic column of YMC-PackODS-A, A mobile phase of acetonitrile (A) and 0.1% phosphoric acid water (B), an ultraviolet detector, A detection wavelength of 203nm, A column temperature of 35 ℃, A flow rate of 1.0m L/min, A sample injection volume of 10 mu L, and A gradient elution program as follows:
| Procedure for measuring the movement of a moving object | Time (min) | Acetonitrile concentration (%) |
| 1 | 0.01 | 15 |
| 2 | 5.00 | 15 |
| 3 | 40.00 | 40 |
| 4 | 90.00 | 80 |
| 5 | 110.00 | 100 |
| 6 | 120.00 | 100 |
step 4, deriving the fingerprints of the 18 batches of blood-activating and pain-relieving capsule test solution obtained in the step 3, introducing the fingerprints into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2004A, selecting chromatographic peaks existing in chromatograms of 18 batches of blood-activating and pain-relieving capsules as common peaks, generating comparison fingerprints of 1 batch of blood-activating and pain-relieving capsules by an average value calculation method, calculating the relative retention time and the relative peak area of each common peak, and as a result, 17 common peaks exist in 1 batch of raw blood-activating and pain-relieving capsules, wherein the comparison fingerprints are shown in figure 1, the fingerprints of 18 batches of test solution are shown in figure 2, the retention time of ginsenoside Rg1 is 21.35min, the retention time of 1 peak in the figure, the retention time of notoginsenoside R1 is 25.62min, the retention time of 3 peak in the figure, the retention time of ginsenoside Re is 27.46min, the retention time of 4 peak in the figure, the retention time of ginsenoside Rb1 is the retention time of No. 5 peak in the figure 39.83min, the retention time of borneol is No. 11 peak in the figure 79.74min, and the retention time of alpha-92.40 peak in the figure 6712 min.
meanwhile, the invention uses the automatically generated reference HP LC fingerprint to generate a common chromatographic peak mode, and the common chromatographic peaks of the 18 batches of the blood-activating and pain-relieving capsule traditional Chinese medicines are analyzed and calculated to have relatively good similarity, which shows that the fingerprint established by the blood-activating and pain-relieving capsule traditional Chinese medicines established by the method can well detect the quality of different batches of the blood-activating and pain-relieving capsules, and the results are shown in Table 2.
TABLE 2 similarity between batches of samples and common patterns
Example 2 forensic study of fingerprint detection methods:
1. Study of precision
the sample serial number S1 test solution prepared according to the method of example 1 is analyzed according to the detection method of example 1, parallel sample injection is carried out for 6 times, the sample injection amount is 10 mu L, the peak area and the retention time of a common peak of a sample HP L C fingerprint are analyzed, the RSD value is calculated by taking alpha-pinene, borneol, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 as reference peaks, the fingerprint comparison and data analysis are carried out by adopting traditional Chinese medicine chromatography similarity evaluation software 2004A, the result similarity is 0.95, and the result shows that the parallel sample injection precision of the device is good.
2. Stability study
the sample serial number S1 test sample solution prepared according to the method of example 1 is analyzed according to the detection method of example 1, sample injection analysis is carried out at different time of 0, 2, 6, 12, 18 and 24 hours, the sample injection amount is 10 mu L, α -pinine, borneol, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 are used as reference peaks, and the similarity is 0.98 by analyzing the peak area and retention time of the common peak of the HP L C fingerprint of the sample and calculating the RSD value, so that the sample solution of the blood-activating and pain-relieving capsule test sample is almost unchanged within 24 hours and has very good stability.
3. Repetitive studies
six samples with the serial number of S1 are precisely weighed in parallel, the weight of the medicine in each blood circulation-promoting and pain-relieving capsule is 1g, 6 parts of the same test solution to be tested are prepared according to the method in the example 1, the sample amount is 10 mu L according to the chromatographic conditions in the example 1, the similarity is 0.97 by analyzing the peak areas and retention time of common peaks of a sample HP L C fingerprint and calculating the RSD value by taking alpha-pinine, borneol, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 as reference peaks, and the result shows that the sample chromatographic peak reproducibility is good and the repeatability of the method is good.
The experimental results show that the fingerprint detection method of the blood-activating and pain-relieving capsule provided by the invention has the advantages of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the blood-activating and pain-relieving capsule, and has important significance for ensuring the clinical curative effect.
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.
Claims (6)
1. A detection method of a fingerprint of a blood circulation promoting and pain relieving capsule is characterized by comprising the following steps:
Step 1, preparation of a blood-activating pain-relieving capsule test solution:
Taking the contents of the blood-activating and pain-relieving capsules of different batches, putting the contents into a conical flask, adding methanol, carrying out ultrasonic treatment, standing, taking supernatant, and filtering the supernatant through a microporous filter membrane to obtain a test solution;
Step 2, preparation of a single-standard reference substance solution:
precisely weighing α -pinene, borneol, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 reference substances, placing in a volumetric flask, adding methanol to constant volume to scale, shaking up, and making into single-standard reference substance solution;
Step 3, precisely absorbing the test solution and the reference solution respectively, injecting the test solution and the reference solution into a high performance liquid chromatograph, and recording a chromatogram;
Step 4, exporting the fingerprint of the blood-activating pain-relieving capsule test solution obtained in the step 3, and introducing the fingerprint into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of blood circulation promoting and pain relieving capsules as common peaks; generating a control fingerprint of the blood-activating and pain-relieving capsule by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the single-standard comparison product solution chromatogram.
2. the method for detecting fingerprint of blood circulation promoting and pain relieving capsule as claimed in claim 1, wherein the step 1 is that the preparation method of the sample solution of blood circulation promoting and pain relieving capsule comprises the steps of taking 18 batches of blood circulation promoting and pain relieving capsules, precisely weighing 0.5g of the content sample of the blood circulation promoting and pain relieving capsule, placing the sample in a 100m L conical flask, adding methanol 500m L, carrying out ultrasonic treatment for 60min, standing, taking the supernatant, and passing through a 0.22 μm microporous filter membrane to obtain the sample solution.
3. the method for detecting fingerprint of blood circulation promoting and pain relieving capsule according to claim 1, wherein the step 2 is a step of preparing a single standard reference solution, wherein the single standard reference solution is prepared by precisely weighing α -pinine, borneol, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rg1 references, placing the reference solutions in a volumetric flask, adding methanol to a constant volume to reach a scale, shaking up, and preparing the single standard reference solutions of the α -pinine of 1.1296mg/m L, the borneol of 0.4136mg/m L, the notoginsenoside R1 of 0.4092mg/m L, the ginsenoside Re of 0.3588mg/m L, the ginsenoside Rb1 of 0.4160mg/m L and the ginsenoside Rg1 of 0.4164mg/m L.
4. the method for detecting the fingerprint of the blood circulation-promoting and pain-relieving capsule according to claim 1, wherein in step 3, the liquid chromatography conditions comprise A chromatographic column of YMC-Pack ODS-A, A mobile phase of acetonitrile A and 0.1% water phosphate B, an ultraviolet detector, A detection wavelength of 203nm, A column temperature of 35 ℃, A flow rate of 1.0m L/min, A sample injection volume of 10 μ L, and A gradient elution procedure as follows:
。
5. The method for detecting fingerprint of blood circulation activating and pain relieving capsule according to claim 1, wherein there are 17 peaks in the fingerprint.
6. the method for detecting fingerprint of blood circulation promoting and pain relieving capsule of claim 1, wherein the retention time of ginsenoside Rg1 is 21.35min and is peak 1, the retention time of notoginsenoside R1 is 25.62min and is peak 3, the retention time of ginsenoside Re is 27.46min and is peak 4, the retention time of ginsenoside Rb1 is 39.83min and is peak 5, the retention time of borneol is 79.74min and is peak 11, and the retention time of α -pinene is 92.40min and is peak 12.
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| CN114839281A (en) * | 2022-04-07 | 2022-08-02 | 珠海安生凤凰制药有限公司 | HPLC (high performance liquid chromatography) -method-based identification method for ground beetles in blood circulation promoting and pain relieving capsules |
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