CN111487335B - Method for detecting fingerprint of Xiao' er Ganmaoshu granules - Google Patents
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Abstract
The invention discloses a method for detecting fingerprint of children's Ganmaoshu granules, which comprises the following steps: step 1, preparing a children cold relieving granule 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, exporting the fingerprint instrument of the Xiao 'er Ganmaoshu granules obtained in the step 3, importing the fingerprint instrument into a traditional Chinese medicine chromatography fingerprint similarity evaluation system, and selecting chromatographic peaks existing in chromatograms of different batches of Xiao' er Ganmaoshu granules as common peaks; generating a control fingerprint of the children Ganmaoshu granules by using an average value calculation method; the relative retention time and the relative peak area of each common peak were calculated. The fingerprint of the Xiao 'er Ganmaoshu granules provided by the invention can comprehensively and objectively represent the quality of the Xiao' er Ganmaoshu granules. 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 medicine, in particular to a detection method of a fingerprint of children's Ganmaoshu granules.
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 children cold relieving granule (national medicine standard character number Z20000076) is a clinical proved prescription of famous paediatrics experts for medical treatment of Jiangyou for decades, consists of eight medicinal materials such as kudzuvine root, great burdock achene, fineleaf schizonepeta herb, platycodon root, figwort root, liquoric root, cicada slough, medicinal fermented mass and the like, and is an effective traditional Chinese medicine preparation for treating exogenous fever of children for clinical use for decades. The formula grasps the etiology of exogenous fever of children and the characteristics of physique of the children, takes pungent and cool radix puerariae as monarch drug, directly clears heat, avoids lung heat, sphagitis and other diseases caused by invasion of heat evil, has the functions of generating yang qi of the child pure yang physique, improves self resistance, drives pathogenic factors to the exterior and prevents the pathogenic factors from entering the interior; in the formula, a large amount of schizonepeta and burdock-seed two exterior-releasing medicines are used as ministerial medicines to eliminate external evil on the body surface, schizonepeta is good at releasing exterior and dispelling wind, and simultaneously has warm characteristics, so that the schizonepeta has the effects of dispelling cold evil and preventing invasion of cold evil, the burdock is good at dispelling wind and dissipating heat and dispelling heat evil on the body surface, and the schizonepeta and the burdock-seed are capable of preventing cold and heat from invading again to cause severe feeling (invasion of secondary external evil); the cicada slough is used together with the pungent and cool exterior-releasing medicine to help the burdock to disperse wind and heat, and meanwhile, the cicada slough has the effects of promoting eruption and relieving itching, so that part of heat pathogen remained in the interior is permeated out by virtue of the yang qi generated by the growth of the children, and the heat pathogen is hidden everywhere in the interior and the exterior; the traditional Chinese medicine considers that the inherent insufficiency of spleen and stomach of children and the lung belong to delicate organs and are extremely easy to be damaged by exogenous pathogenic factors, so that the viscera such as the spleen, the stomach, the lung and the like of children must be damaged after the cold is caused, and the traditional Chinese medicine is very important for regulating the spleen and the stomach, clearing away the lung-heat, reducing phlegm and tonifying vital qi; the figwort root is selected in the formula for nourishing yin and moistening dryness to prepare the kudzuvine root to grow redundant yang qi, so that excessive yang qi is prevented from causing excessive internal heat, and the yin-yang balance is achieved; the Jianqu is selected to strengthen the stomach and promote digestion so as to recuperate the lesion and the lesion of the spleen and the stomach; the platycodon grandiflorum ascends the clear and descends the turbid, enters lung channels, disperses the lung and relieves sore throat, eliminates phlegm and well conditions the damaged lung; the prescription takes the liquorice as a guiding drug, has the effects of tonifying middle-jiao and Qi and moderating the property of the drug, and ensures that the drug gradually reaches the focus of infection so as to increase the property of the drug. The whole formula closely follows the characteristics of physical characteristics of children and the pathological development of the exogenous cold of the children as prescription basis, takes the removal of exogenous pathogenic factors and the conditioning of physical health as guiding principles, not only well treats the disease, but also conditions the body, resists the invasion of exogenous pathogenic factors, and really realizes the treatment of both principal and secondary aspects of diseases caused by the exogenous pathogenic factors of the children.
At present, no fingerprint detection method about the children cold soothing granules is reported, and in order to objectively, comprehensively and accurately evaluate the quality of the children cold soothing granules, control and guarantee the quality of the children cold soothing granules and guarantee the clinical curative effect, it is necessary to develop a fingerprint spectrum research on the basis of the prior art.
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 children's Ganmaoshu granules, which can objectively, comprehensively and accurately evaluate the quality of the children's Ganmaoshu granules and has important significance for controlling the quality of the children's Ganmaoshu granules 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 fingerprint of Xiao' er Ganmaoshu granule for treating common cold comprises the following steps:
step 1, preparing a children cold relieving granule test solution:
respectively and precisely weighing infantile common cold relieving granule samples of different batches of infantile common cold relieving granules, placing in a conical flask, adding methanol, performing ultrasonic treatment, filtering, collecting filtrate, volatilizing in water bath, adding methanol for dissolving, fixing volume, and filtering with 0.22 μm microporous membrane to obtain test solution;
precisely weighing puerarin, chlorogenic acid, liquiritin, arctiin and harpagoside reference substances, placing in a volumetric flask, fixing the volume to scale with methanol, shaking up, and making into single-standard reference substance solution;
step 4, exporting the fingerprint of the infantile common cold Shu 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 the children cold relieving granules as common peaks; generating a control fingerprint of the children Ganmaoshu granules 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.
As a preferred scheme, in the method for detecting the fingerprint of the Xiao 'er Ganmaoshu granule, step 1, the method for preparing the test solution of the Xiao' er Ganmaoshu granule comprises the following steps: taking 12 batches of the infantile common cold relieving granules, precisely weighing 5g of a sample of the infantile common cold relieving granules, putting the sample into a 50mL conical flask, adding 30mL of methanol, carrying out ultrasonic treatment for 30min, filtering, volatilizing filtrate in water bath, dissolving the volatilized matter with the methanol, fixing the volume to 25mL, then taking 1mL of each sample solution, fixing the volume to 5mL, and filtering through a 0.22-micron microporous filter membrane to obtain a sample solution.
As a preferred scheme, the method for detecting the fingerprint of the Xiao' er Ganmaoshu granule comprises the following steps of 2: accurately weighing puerarin, chlorogenic acid, liquiritin, arctiin and harpagoside reference substances, placing in a volumetric flask, fixing the volume to scale with methanol, shaking uniformly, and preparing into 75.02 μ g/mL puerarin, 79.22 μ g/mL chlorogenic acid, 77.67 μ g/mL liquiritin, 76.23 μ g/mL arctiin and 59.23 μ g/mL harpagoside single-standard reference substance solution.
As a preferred scheme, in the method for detecting the fingerprint of the Xiao' er Ganmaoshu granule, in step 3, the liquid chromatography conditions are as follows: a chromatographic column: YMC-Pack ODS-A, mobile phase: acetonitrile and water, SPD-20A detector, detection wavelength: 280nm, column temperature 40 ℃, flow rate 1.0mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:
| Procedure | time/min | Acetonitrile volume (%) |
| 1 | 0.01 | 5 |
| 2 | 5.00 | 5 |
| 3 | 15.00 | 5 |
| 4 | 35.00 | 10 |
| 5 | 55.00 | 30 |
| 6 | 60.00 | 60 |
| 7 | 62.00 | 100 |
| 8 | 65.00 | 100 |
As a preferred scheme, in the method for detecting the fingerprint of the Xiao' er Ganmaoshu granule, 12 common peaks are present in the fingerprint.
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;
the flow rates (1mL/min, 0.8mL/min, 0.7mL/min, 0.6mL/min and 0.5mL/min) are screened, and because isomers and other components with extremely similar polarities exist in the components in the children Ganmaoshu granules, 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 the gradient condition that the flow rate is 1mL/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. The result shows that when acetonitrile and water are used as the mobile phase, the components in the children cold relieving granule can achieve good separation effect, so that acetonitrile and water are finally selected as the mobile phase.
After the optimal mobile phase is determined, in order to obtain better separation degree and establish a fingerprint spectrum with a plurality of common peaks with good separation degree, the invention screens a large number of elution modes, and finally determines the optimal elution mode as follows: 0.01-5 min, and 5% of acetonitrile; 5-15 min, and 5% of acetonitrile; 15-35 min, 10% of acetonitrile; 35-55 min and 30% of acetonitrile; 55-60 min, and 60% of acetonitrile; 60-62 min, and 100% of acetonitrile; 62-65 min and 100% of acetonitrile.
Has the beneficial effects that:
1. according to the structural property characteristics of active ingredients contained in the children's cold comforting granules, the optimal mobile phase composition is screened out through a large number of experiments, 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, and the fingerprint spectrum detection method for the children's cold comforting granules can comprehensively, objectively and accurately detect and evaluate the quality of the children's cold comforting granules and has important significance for ensuring the curative effect of the clinical children's cold comforting granules.
2. The fingerprint of the Xiao ' er Ganmaoshu granules established by the method provided by the invention can effectively represent the quality of the Xiao ' er Ganmaoshu granules, objectively reflect the front and back sequence and the mutual relation of each formed fingerprint characteristic peak, pay attention to the overall facial features, avoid the one-sidedness of the quality of the Xiao ' er Ganmaoshu granules determined by measuring individual chemical components, and reduce the possibility of manual treatment for reaching the quality standard.
3. The method for detecting the fingerprint of the children's cold soothing granules 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 the Xiao' er Ganmaoshu granule sample of the present invention.
Fig. 2 is a fingerprint of 12 batches of the test article of the infantile Ganmaoshu 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 corporation in Japan comprises a full-automatic online degassing system, a full-automatic sample injection system SIL-20A, an ultraviolet detector SPD-20A, an automatic temperature control column temperature box CTD-20AC, KH-500E type ultrasonic cleaner (Kunshan Seama ultrasonic instruments Co., Ltd.), and an ML104/02 electronic analytical balance (Mettler Toledo).
1.2 drugs and reagents
The source of the infantile common cold particle sample (national standard medicine Z20000076) is shown in Table 1; puerarin (batch No. 110752-); chlorogenic acid (batch No. 110753-201817); glycyrrhizin (batch No. 111610-201908); the harpagoside (batch No. 111730-2018508) is purchased from China institute for testing food and drug; arctiin (batch No. 110819-; methanol (analytically pure); acetonitrile (chromatographically pure); water (Wahaha purified water).
TABLE 1 infantile GANMAOSHU granule sample information table
| Sample number | Batch number | Manufacturer of the product |
| S1 | 171101 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S2 | 171102 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S3 | 171103 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S4 | 171104 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S5 | 180401 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S6 | 180402 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S7 | 180403 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S8 | 180404 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S9 | 200101 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S10 | 200102 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S11 | 200103 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
| S12 | 200104 | NANJING ZHONGSHAN PHARMACEUTICAL Co.,Ltd. |
。
Embodiment 1 a method for detecting fingerprint of Xiao' er Ganmaoshu granule, comprising the following steps:
step 1, preparing a children cold relieving granule test solution:
taking 12 batches of the infantile common cold relieving granules in the table 1, precisely weighing 5g of a sample of the infantile common cold relieving granules respectively, placing the sample in a 50mL conical flask, adding 30mL of methanol, carrying out ultrasonic treatment for 30min, filtering, volatilizing filtrate in water bath, dissolving the volatilized matter with the methanol, fixing the volume to 25mL, then taking 1mL of each sample solution, fixing the volume to 5mL, and filtering through a 0.22 mu m microporous filter membrane to obtain a sample solution.
accurately weighing puerarin, chlorogenic acid, liquiritin, arctiin and harpagoside reference substances, placing in a volumetric flask, fixing the volume to scale with methanol, shaking uniformly, and preparing 75.02 mu g/mL puerarin, 79.22 mu g/mL chlorogenic acid, 77.67 mu g/mL liquiritin, 76.23 mu g/mL arctiin and 59.23 mu g/mL harpagoside reference substance solution.
| Procedure | time/min | Acetonitrile volume (%) |
| 1 | 0.01 | 5 |
| 2 | 5.00 | 5 |
| 3 | 15.00 | 5 |
| 4 | 35.00 | 10 |
| 5 | 55.00 | 30 |
| 6 | 60.00 | 60 |
| 7 | 62.00 | 100 |
| 8 | 65.00 | 100 |
Step 4, exporting the fingerprints of the 12 batches of the infantile common cold particles to-be-tested sample solutions obtained in the step 3, and importing the fingerprints into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of 12 batches of the children cold relieving granules as common peaks; generating a control fingerprint of 1 batch of the children Ganmaoshu granules by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; as a result, 12 common peaks were observed in 1 batch of crude Xiaotonganmaoshu granules, and the reference fingerprint is shown in FIG. 1, and the fingerprint of 12 batches of the test sample is shown in FIG. 2. Through comparison and analysis of the reference substances, the puerarin retention time is 21.620min, the chlorogenic acid retention time is 3.815min, the liquiritin retention time is 28.393min, the arctiin retention time is 39.755min, and the harpagoside retention time is 41.751 min.
Meanwhile, the invention uses the automatically generated reference HPLC fingerprint R to generate a common chromatographic peak mode, and relatively good similarity is obtained among 12 batches of common chromatographic peaks of the children cold relieving granule traditional Chinese medicines through analysis and calculation, which shows that the fingerprint established by the children cold relieving granule traditional Chinese medicines established by the method can well detect the quality of different manufacturers and batches of children cold relieving granules, and the results are shown in Table 2.
TABLE 2 similarity between batches of samples and common patterns
Example 2 Law study of fingerprint detection method
1. Study of precision
The sample serial number prepared according to the method of example 1 is S1 test solution, the detection method of example 1 is used for analysis, parallel sample injection is carried out for 6 times, the sample injection amount is 10 mu L, chlorogenic acid, puerarin, liquiritin, arctiin and harpagoside are used as reference peaks, the peak area and retention time of common peaks of HPLC fingerprint of the sample are analyzed, RSD values are calculated, the result is shown in table 3, fingerprint comparison and data analysis are carried out by adopting Chinese medicine chromatographic similarity evaluation software 2004A, the result similarity is 0.96, and the result shows that the parallel sample injection precision of the device is good.
TABLE 3 precision study of peak area and retention time
2. Stability study
The sample serial number of the sample solution prepared according to the method of example 1 is S1 test sample solution, the detection method of example 1 is adopted, the sample injection analysis is performed at different time periods of 0 hour, 2 hours, 6 hours, 12 hours, 18 hours and 24 hours, the sample injection amount is 10 μ L, chlorogenic acid, puerarin, liquiritin, arctiin and harpagoside are used as reference peaks, the result is shown in table 4, the similarity is 0.97, and the result shows that the chromatographic peak of the test sample solution of the Xiao' er Ganmaoshu granule is almost unchanged within 24 hours and the stability is very good by analyzing the peak area and the retention time of the common peaks of the sample HPLC fingerprint and calculating the RSD value.
Table 4 stability study peak area and retention time
3. Repetitive studies
Six samples with the serial number of S1 are weighed in parallel and accurately, the weight of each part of the Xiaoer Ganmaoshu granule traditional Chinese medicine is 5g, 6 parts of the same test solution is prepared according to the method in the embodiment 1, the sample injection amount is 10 mu L according to the chromatographic condition in the embodiment 1, chlorogenic acid, puerarin, liquiritin, arctiin and harpagoside are used as reference peaks, the result is shown in a table 5, the similarity is 0.95, the result shows that the repeatability of the sample chromatographic peak is good and the repeatability of the method is good by analyzing the peak area and the retention time of the common peak of the sample HPLC fingerprint and calculating the RSD value.
TABLE 5 repeated study of peak area and retention time
The experimental results show that the fingerprint spectrum detection method for the Xiao 'er Ganmaoshu granules provided by the invention has the advantages of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the Xiao' er Ganmaoshu granules, 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 (4)
1. A method for detecting a fingerprint of Xiao' er Ganmaoshu granules is characterized by comprising the following steps:
step 1, preparing a children cold relieving granule test solution:
respectively and precisely weighing infantile common cold relieving granule samples in different batches, placing in a conical flask, adding methanol, performing ultrasonic treatment, filtering, collecting filtrate, volatilizing in water bath, adding methanol for dissolving, diluting to desired volume, and filtering with microporous membrane to obtain sample solution;
step 2, preparation of a reference solution:
accurately weighing puerarin, chlorogenic acid, liquiritin, arctiin and harpagoside reference substances, placing in a volumetric flask, adding methanol to constant volume to scale, shaking up, and making into mixed reference substance solution;
step 3, respectively and precisely absorbing the test solution and the reference solution, 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 infantile common cold Shu 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 the children cold relieving granules as common peaks; generating a control fingerprint of the children Ganmaoshu granules by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the single-standard comparison product solution chromatogram;
in step 3, the liquid chromatography conditions are as follows: a chromatographic column: YMC-Pack ODS-A, mobile phase: acetonitrile and water, ultraviolet detector, detection wavelength: 280nm, column temperature 40 ℃, flow rate 1.0mL/min, sample injection volume: 10 μ L, gradient elution procedure as follows:
。
2. The method for detecting the fingerprint of the Xiao 'er Ganmaoshu granules according to claim 1, wherein the preparation method of the test solution of the Xiao' er Ganmaoshu granules in step 1 is as follows: taking 12 batches of the infantile common cold relieving granules, precisely weighing 5g of a sample of the infantile common cold relieving granules, putting the sample into a 50mL conical flask, adding 30mL of methanol, carrying out ultrasonic treatment for 30min, filtering, volatilizing filtrate in water bath, dissolving the volatilized matter with the methanol, fixing the volume to 25mL, then taking 1mL of each sample solution, fixing the volume to 5mL, and filtering through a 0.22-micron microporous filter membrane to obtain a sample solution.
3. The method for detecting the fingerprint of Xiao' er Ganmaoshu granules according to claim 1, wherein the step 2 is to prepare a reference solution: accurately weighing puerarin, chlorogenic acid, liquiritin, arctiin and harpagoside reference substances, placing in a volumetric flask, fixing the volume to scale with methanol, shaking up, and preparing into 75.02 μ g/mL puerarin, 79.22 μ g/mL chlorogenic acid, 77.67 μ g/mL liquiritin, 76.23 μ g/mL arctiin and 59.23 μ g/mL harpagoside mixed reference substance solution.
4. The method for detecting the fingerprint of Xiao' er Ganmaoshu granules according to claim 1, wherein there are 12 common peaks in the fingerprint.
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| HPLC法测定小儿感冒颗粒中绿原酸、木犀草苷3,5-O-二咖啡酰基奎宁酸、连翘苷的含量;任菲菲 等;《中国药品标准》;20191231;第20卷(第4期);第325-331页 * |
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