CN111487344A - Method for detecting fingerprint spectrum of motherwort particles - Google Patents
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Abstract
The invention discloses a method for detecting fingerprint spectrum of motherwort particles, which comprises the following steps: step 1, preparing a motherwort granule test solution; step 2, preparation of a mixed reference 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 motherwort particles obtained in the step 3, leading the fingerprint instrument into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and selecting chromatographic peaks existing in chromatograms of different batches of motherwort particles as common peaks; generating a control fingerprint of the motherwort particles by using an average calculation method; the relative retention time and the relative peak area of each common peak were calculated. The fingerprint of the motherwort particles provided by the invention can comprehensively and objectively characterize the quality of the motherwort particles. 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 traditional Chinese medicines, in particular to a detection method of fingerprint spectrums of motherwort particles.
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.
Herba Leonuri granule (national medicine standard No. Z32020148) is prepared by extracting herba Leonuri with water, concentrating into extract, adding sucrose powder and appropriate amount of dextrin, mixing, and making into granule. Has effects of promoting blood circulation and regulating menstruation, and can be used for treating menoxenia due to blood stasis with symptoms of small menstrual flow and dripping. After the product is subjected to an extraction and concentration preparation process, the contained chemical components are changed, new effective components of 5-hydroxymethylfurfural are generated, and part of unknown peaks are generated.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art and provides a fingerprint detection method for motherwort particles, which can objectively, comprehensively and accurately evaluate the quality of the motherwort particles and has important significance for controlling the quality of the motherwort particles 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 method for detecting a fingerprint spectrum of motherwort particles is characterized by comprising the following steps:
step 1, preparation of a motherwort granule test solution:
taking motherwort particles of different batches, respectively and precisely weighing motherwort particle samples, putting the motherwort particle samples into a conical flask, adding methanol, carrying out constant-temperature ultrasonic treatment, taking supernate, volatilizing the supernate in water bath, adding methanol for dissolving, fixing the volume, and filtering the supernate with a microporous filter membrane to obtain a test solution;
precisely weighing 5-hydroxymethylfurfural, rutin, leonurine hydrochloride and hyperin reference substance, placing in a volumetric flask, fixing the volume to a scale with methanol, shaking up to obtain a mixed reference substance solution;
Preferably, the above-mentioned method for detecting fingerprint of herba Leonuri granule comprises, in step 1, preparing a sample solution of herba Leonuri granule by collecting 9 batches of herba Leonuri granule, precisely weighing 3g of herba Leonuri granule sample, placing in a 50m L conical flask, adding 25m L methanol, subjecting to ultrasonic treatment at 40 deg.C for 60min, collecting the supernatant of 10m L, volatilizing in water bath, dissolving the volatilized matter with methanol to a volume of 2m L, and filtering with 0.22 μm microporous membrane to obtain the sample solution.
Preferably, the method for detecting fingerprint of herba leonuri particle comprises the step 2 of preparing a mixed reference solution, wherein the mixed reference solution is prepared by taking a precisely-weighed 5-hydroxymethylfurfural, rutin, leonurine hydrochloride and hyperoside reference, placing the reference solution in a volumetric flask, fixing the volume to scale with methanol, shaking up, and preparing the mixed reference solution of 57.49 mu g/m L of 5-hydroxymethylfurfural, 80.64 mu g/m L of rutin, 74.58 mu g/m L of leonurine hydrochloride and 74.50 mu g/m L of hyperoside.
Preferably, in the method for detecting fingerprint of motherwort herb particles, in step 3, the liquid chromatography conditions comprise A chromatographic column of YMC-Pack ODS-A, A mobile phase of acetonitrile and 0.1% phosphoric acid water, an ultraviolet detector, A detection wavelength of 280nm, 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 volume (%) |
| 1 | 0.01 | 5 |
| 2 | 3.00 | 5 |
| 3 | 50.00 | 25 |
| 4 | 65.00 | 50 |
| 5 | 85.00 | 100 |
| 6 | 90.00 | 100 |
Preferably, in the method for detecting fingerprint of leonurus japonicus granules, 16 peaks are shared in the fingerprint. Wherein the retention time of 5-hydroxymethylfurfural is 26.678min, peak 5 in the figure, the retention time of rutin is 38.867min, peak 9 in the figure, the retention time of leonurine hydrochloride is 41.520min, peak 10 in the figure, the retention time of hyperin is 33.590min, and peak 6 in the figure.
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 254nm and 280nm are extracted, and when the detection wavelength is 280nm, the information content contained in the chromatogram maps is most comprehensive and the base line is stable, so that 280nm is selected as the detection wavelength;
in addition, 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 the components in the motherwort particles mostly contain isomers and other components with very similar polarities, the result shows that when acetonitrile and 0.1% phosphoric acid water are used as a mobile phase, each component in the motherwort particles can achieve a good separation effect, so that the acetonitrile and the 0.1% phosphoric acid water are finally selected as the mobile phase.
In addition, the invention screens the flow velocity (1m L/min, 0.8m L/min, 0.7m L/min, 0.6m L/min and 0.5m L/min), and finds that the separation can not be carried out at high flow velocity, so that the separation effect is better at low flow velocity, and finally the substances with similar polarity are separated under the gradient conditions of multiple times of flow velocity of 1m L/min and the like.
Has the advantages that:
1. according to the structural property characteristics of active ingredients contained in the motherwort particles, 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 motherwort grains established by the method provided by the invention can effectively represent the quality of motherwort grains, objectively reflect the front and back sequence and the mutual relation of characteristic peaks of each formed fingerprint, pay attention to the overall feature, avoid the one-sidedness of judging the quality of motherwort grains 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 motherwort particles provided by the invention has the advantages of simplicity, convenience, good stability, high precision, good reproducibility and the like.
Drawings
FIG. 1 is a comparison fingerprint of a sample of motherwort herb granules of the present invention.
FIG. 2 shows the fingerprint of 9 batches of the motherwort herb granule sample.
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, Japan, comprises a full-automatic online degassing system, a full-automatic sample feeding system SI L-20A, an ultraviolet detector SPD-20A, an automatic temperature control column temperature box CTD-20AC, KH-500E type ultrasonic cleaner (Kunshan Seawa ultrasonic instruments, Ltd.), a DK-98-IIA electric heating constant temperature water bath (Tensted instruments, Tianjin), and an M L104/02 electronic analysis balance (Mettler Toledo).
1.2 drugs and reagents
The sample sources of the motherwort particles are shown in table 1; rutin (batch No. 0080-9705) was purchased from China pharmaceutical biologicals institute; 5-hydroxymethylfurfural (batch No. 111626-; hyperin (batch No. 111521-201507) was purchased from the institute of food and drug testing; methanol (analytically pure); phosphoric acid (analytically pure); acetonitrile (chromatographically pure); water (Wahaha purified water).
TABLE 1 motherwort granule sample information table
Embodiment 1 a method for detecting fingerprint of motherwort particles, comprising the following steps:
step 1, preparation of a motherwort granule test solution:
taking 9 batches of motherwort particles in the table 1, precisely weighing 3g of motherwort particle samples respectively, placing the samples in 50m L conical flasks, adding 25m L of methanol respectively, carrying out ultrasonic treatment at the water temperature of 40 ℃ for 60min, taking 10m L of supernate, volatilizing the supernate in a water bath, dissolving the volatilized substances in the methanol, fixing the volume to 2m L, and filtering the solution through a 0.22-micron microporous filter membrane to obtain 9 batches of test solution.
precisely weighing 5-hydroxymethylfurfural, rutin, leonurine hydrochloride and hyperin reference substances, placing the reference substances in a volumetric flask, fixing the volume to scale by using methanol, shaking up uniformly, and preparing a mixed reference substance solution of 57.49 mu g/m L of 5-hydroxymethylfurfural, 80.64 mu g/m L of rutin, 74.58 mu g/m L of leonurine hydrochloride and 74.50 mu g/m L of hyperin.
And 3, precisely sucking 9 batches of leonurus particle test solution and reference solution prepared in the step 1 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-Pack ODS-A, A mobile phase of acetonitrile and 0.1% phosphoric acid water, an ultraviolet detector, A detection wavelength of 280nm, 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 are as follows:
| procedure for measuring the movement of a moving object | Time (min) | Acetonitrile volume (%) |
| 1 | 0.01 | 5 |
| 2 | 3.00 | 5 |
| 3 | 50.00 | 25 |
| 4 | 65.00 | 50 |
| 5 | 85.00 | 100 |
| 6 | 90.00 | 100 |
Meanwhile, the invention uses the automatically generated reference HP L C fingerprint R to generate a common chromatographic peak mode, and relatively good similarity among common chromatographic peaks of 9 batches of motherwort particle traditional Chinese medicines is obtained through analysis and calculation, which shows that the fingerprint established by the motherwort particle traditional Chinese medicines established by the method can well detect the quality of different batches of motherwort particles, and the result is 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, 5-hydroxymethyl furfural, rutin, leonurine hydrochloride and hyperoside are used as reference peaks, the RSD value is calculated by analyzing the peak area and retention time of a common peak of a sample HP L C fingerprint, the result is shown in table 3, fingerprint comparison and data analysis are carried out by adopting Chinese medicine chromatography similarity evaluation software 2004A, the result similarity is 0.99, and the result shows that the precision of parallel sample injection of the device is good.
TABLE 3 precision study of peak area and retention time
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, 5-hydroxymethyl furfural, rutin, leonurine hydrochloride and hyperoside are used as reference peaks, the RSD value is calculated by analyzing the peak area and retention time of a common peak of a HP L C fingerprint of the sample and the result is shown in Table 4, the similarity is 0.95, and the result shows that the chromatographic peak of the leonurus particle test sample solution in 24 hours is almost unchanged and the stability is very good.
Table 4 stability study peak area and retention time
3. Repetitive studies
Six samples which are numbered as S1 are precisely weighed in parallel, the weight of each motherwort particle traditional Chinese medicine is 5g, 6 parts of the same test solution to be tested are prepared according to the method in the embodiment 1, the sample amount is 10 mu L according to the chromatographic conditions in the embodiment 1, 5-hydroxymethyl furfural, rutin, leonurine hydrochloride and hyperoside are used as reference peaks, the RSD value is calculated by analyzing the peak area and the retention time of the common peak of the HP L C fingerprint of the sample and the result is shown in the table 5, the similarity is 0.95, and the result shows that the sample chromatographic peak reproducibility is good and the repeatability of the method is good.
TABLE 5 repeated study of peak area and retention time
The experimental results show that the leonurus particle fingerprint spectrum detection method provided by the invention has the advantages of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of leonurus particles, and has important significance for ensuring 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 method for detecting a fingerprint spectrum of motherwort particles is characterized by comprising the following steps:
step 1, preparation of a motherwort granule test solution:
taking motherwort particles of different batches, respectively and precisely weighing motherwort particle samples, putting the motherwort particle samples into a conical flask, adding methanol, carrying out constant-temperature ultrasonic treatment, taking supernate, volatilizing the supernate in water bath, adding methanol for dissolving, fixing the volume, and filtering the supernate with a microporous filter membrane to obtain a test solution;
step 2, preparation of a mixed reference solution:
precisely weighing 5-hydroxymethylfurfural, rutin, leonurine hydrochloride and hyperin reference substances, putting the reference substances into a volumetric flask, fixing the volume to a scale with methanol, and shaking up to prepare a mixed 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 motherwort granule test solution obtained in the step 3, and importing the fingerprint into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of motherwort particles as common peaks; generating a control fingerprint of the motherwort particles 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 mixed comparison product solution chromatogram.
2. The method for detecting the fingerprint of motherwort particles as claimed in claim 1, wherein the preparation method of the motherwort particle sample solution in step 1 comprises the steps of taking 9 batches of motherwort particles, precisely weighing 3-9 g of motherwort particle samples, placing the motherwort particle samples in a 50-150 m L conical flask, adding 25-75 m L of methanol, carrying out ultrasonic treatment at 40 ℃ for 60min, taking 10-30 m L of supernate, volatilizing the samples in a water bath, dissolving the volatilized substances with the methanol, fixing the volume to 2-6 m L, and filtering the samples with a 0.22 μm microporous filter membrane to obtain the sample solution.
3. The method for detecting the fingerprint of motherwort herb granules according to claim 1, wherein the preparation of the mixed reference solution in the step 2 comprises the steps of precisely weighing 5-hydroxymethylfurfural, rutin, leonurine hydrochloride and hyperoside reference, putting the weighed reference in a volumetric flask, fixing the volume to the scale with methanol, shaking up, and preparing the mixed reference solution of 57.49 μ g/m L of 5-hydroxymethylfurfural, 80.64 μ g/m L of rutin, 74.58 μ g/m L of leonurine hydrochloride and 74.50 μ g/m L of hyperoside.
4. The method for detecting fingerprint of motherwort herb particles as claimed in claim 1, wherein in step 3, the liquid chromatography conditions are YMC-Pack ODS-A, mobile phase is acetonitrile and 0.1% phosphoric acid water, gradient elution is performed, ultraviolet detector is performed, detection wavelength is 280nm, column temperature is 35 ℃, flow rate is 1.0m L/min, sample injection volume is 10 μ L, and gradient elution program is as follows:
。
5. The method for detecting the fingerprint of motherwort particles according to claim 1, wherein there are 16 common peaks in the fingerprint.
6. The motherwort herb particle fingerprint detection method according to claim 5, wherein the retention time of 5-hydroxymethylfurfural is 26.678min, which is peak 5; the retention time of rutin is 38.867min, which is No. 9 peak; the retention time of the leonurine hydrochloride is 41.520min, which is No. 10 peak; the retention time of hyperin was 33.590min, peak 6.
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| CN112946134A (en) * | 2021-02-04 | 2021-06-11 | 成都第一制药有限公司 | HPLC fingerprint spectrum of motherwort injection and establishing method thereof |
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| CN114518338A (en) * | 2022-02-07 | 2022-05-20 | 吉林省现代中药工程研究中心有限公司 | Construction and application of method for rapidly detecting quality of motherwort particles |
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| CN112946134A (en) * | 2021-02-04 | 2021-06-11 | 成都第一制药有限公司 | HPLC fingerprint spectrum of motherwort injection and establishing method thereof |
| CN114235996A (en) * | 2021-12-06 | 2022-03-25 | 广东一方制药有限公司 | Fresh motherwort herb, standard decoction thereof and traditional Chinese medicine formula particle characteristic spectrum construction and detection method |
| CN114518338A (en) * | 2022-02-07 | 2022-05-20 | 吉林省现代中药工程研究中心有限公司 | Construction and application of method for rapidly detecting quality of motherwort particles |
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