CN115436527A

CN115436527A - Juanbi soup substance reference HPLC fingerprint spectrum and determination method

Info

Publication number: CN115436527A
Application number: CN202211204954.5A
Authority: CN
Inventors: 罗宇东; 陈晓艺; 韦雯怡; 谭安蔷; 邹意华
Original assignee: Guangxi University Of Traditional Chinese Medicine Bainianle Pharmaceutical Co ltd
Current assignee: Guangxi University Of Traditional Chinese Medicine Bainianle Pharmaceutical Co ltd
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2022-12-06

Abstract

The invention provides a substance-based HPLC fingerprint spectrum for Juanbi decoction and a determination method, belonging to the technical field of traditional Chinese medicine analysis. The method comprises the following steps: s1, preparing a reference substance solution; s2, preparing a test solution; s3, carrying out chromatographic conditions; and S4, measuring. The fingerprint spectrum measuring method of Juanbi soup material standard has strong specificity, better reproducibility and stability, simple method and better aim of controlling the internal quality of the product, so the fingerprint spectrum measuring method of Juanbi soup material standard is used as the quality control method of the product.

Description

Juanbi soup substance reference HPLC fingerprint spectrum and determination method

Technical Field

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a Juanbi soup substance standard HPLC fingerprint spectrum and a determination method.

Background

Arthralgia-treating decoction comes from the chapter of Bing san Bi (arthroncus of knee) from Yi Shu san from Yi Xue Xin Gong, chi Dynasty. Treating arthralgia syndrome by combining the three qi of wind, cold and dampness. Comprises one part of notopterygium root and radix angelicae pubescentis, one part of cassia bark, five parts of gentiana macrophylla, three parts of angelica sinensis, seven parts of ligusticum wallichii, five parts of liquorice (roasted), two parts of kadsura pepper stem, three parts of mulberry twig, frankincense and eight parts of elecampane. It can be used for treating apoplexy, vexation, pain, spasm of neck and back, cold limbs, arthralgia, heaviness of waist and knee, and difficulty in movement. In the formula, notopterygium root, radix angelicae pubescentis, gentiana macrophylla and cinnamon are used for expelling wind, removing cold and eliminating dampness; angelica, rhizoma ligustici wallichii and frankincense have the effects of regulating qi and nourishing blood, and activating blood and relieving pain; ramulus Mori and caulis Piperis Futokadsurae have the effects of dredging meridian passage and relieving pain. Modern pharmacological research proves that: the water soluble component of Notopterygii rhizoma can inhibit delayed type allergy and inflammatory reaction; the radix Angelicae Pubescentis water extract has antiinflammatory and analgesic effects; ferulic acid in radix Angelicae sinensis and rhizoma Ligustici Chuanxiong has effects of enhancing blood supply of myocardium and relieving myocardial ischemia; the gentiana macrophylla has the effects of resisting inflammation, easing pain and relieving heat, indirectly influences pituitary by a neurohumoral system, promotes the enhancement of adrenal cortex function and the increase of corticoid secretion; the whole formula has the effects of resisting inflammation, easing pain, regulating immunity and improving microcirculation, thereby increasing blood and oxygen supply of nerve tissues and promoting repair of peripheral nerve injury, is a common prescription for clinically treating anemofrigid-damp arthralgia, and has the effects of dispelling wind and cold, and eliminating dampness and dredging arthralgia.

Bi Tang belongs to the category of 79 from 100 classic famous parties in the list of ancient classic famous parties (first group) formulated by the national State administration of medicine and health supervision in 2018. The classic famous prescription is the standard of the traditional Chinese medicine medicinal substance prepared according to the preparation method of the ancient classic famous prescription recorded by the ancient medical books, and except for the molding process, the other preparation methods are basically consistent with the records of the ancient medical books. The forming process generally adopts a mode of not damaging the composition of the material, such as freeze drying, spray drying and the like. The clinical curative effect of the classic famous prescription is exact, the classic famous prescription is developed and carried with a traditional Chinese medicine preparation which is convenient to carry and take by applying modern scientific technology, and the traditional Chinese medicine preparation is not only passed with traditional Chinese medicines, but also can better promote the clinical application of the classic famous prescription. The granule preparation has the advantages of simple preparation process, easy shaping, convenient administration and carrying, and the administration form is most consistent with that of the traditional decoction, and the advantages of the traditional decoction can be retained to a great extent, so the granule is most suitable for the classical famous prescription taking in the form of decoction.

The traditional Chinese medicine fingerprint is an important analysis means for controlling the quality of the traditional Chinese medicine, and can ensure the internal quality of the traditional Chinese medicine to be uniform and stable. Through research and application for over ten years, the technology is mature day by day and is widely applied to the control of the quality standard of the traditional Chinese medicine. The preparation and application of the traditional Chinese medicine fingerprint usually comprise: (1) Collecting representative traditional Chinese medicine samples, preparing a test solution by adopting a proper method, analyzing a certain batch of traditional Chinese medicine samples according to a determined analysis method, and processing the obtained multiple batches of data to obtain a standard fingerprint; (2) After the sample is processed and separated according to the method same as the standard fingerprint spectrum, the similarity of the obtained fingerprint spectrum and the standard fingerprint spectrum is compared, and the sample can be regarded as qualified when the similarity is more than 0.9. At present, few finger print documents about Juanbi decoction exist, and no report is made about the component analysis and quality control of the formula. Therefore, the method has great development value. In order to more comprehensively and effectively control the quality of the clinical medication of the Juanbi decoction preparation and ensure the safety and the curative effect of the Juanbi decoction preparation, a more advanced quality detection means needs to be adopted for the Juanbi decoction composition to achieve the aim of effectively controlling the quality of the Juanbi decoction preparation product.

Disclosure of Invention

The invention aims to provide a Juanbi soup substance-based HPLC fingerprint and a determination method, wherein the Juanbi soup substance-based fingerprint determination method has the advantages of strong specificity, better reproducibility and stability, simple and convenient method and better control of the internal quality of the product, so that the Juanbi soup substance-based fingerprint determination is used as the quality control method of the product.

The technical scheme of the invention is realized as follows:

the invention provides a method for measuring standard HPLC fingerprint spectrum of Juanbi decoction material, which comprises the following steps:

s1, preparation of a reference substance solution: precisely weighing appropriate amount of chlorogenic acid, gentiopicrin, ferulic acid, liquiritin and senkyunolide I, dissolving in methanol, and respectively making into mixed reference solution;

s2, preparing a test solution: precisely weighing the Juanbi decoction, placing in a volumetric flask, adding methanol to constant volume, ultrasonically extracting, cooling to room temperature, supplementing lost weight, shaking, and filtering to obtain a test solution;

s3, chromatographic conditions: adopting an AgilentZORBAXSB-C18 column, wherein the mobile phase A is acetonitrile, the mobile phase B is 0.05-0.15wt% formic acid solution, adopting gradient elution, the flow rate is 0.5-1.5ml/min, the column temperature is 28-32 ℃, and the detection wavelength is as follows: 280-320nm; the sample injection amount is 5-15 mul;

s4, determination: precisely absorbing the reference solution and the test solution respectively, injecting into a high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step S3.

As a further improvement of the invention, the concentrations of chlorogenic acid, gentiopicrin, ferulic acid, liquiritin and senkyunolide I in the mixed reference solution in the step S1 are 0.19-0.21mg/ml, 0.09-0.11mg/ml and 0.09-0.11mg/ml.

As a further improvement of the invention, the ultrasonic extraction time in the step S2 is 30-50min, the power is 180-220W, and the frequency is 35-45kHz.

As a further improvement of the invention, in the step S2, the ultrasonic extraction time is 40min, the power is 200W, and the frequency is 40kHz.

As a further improvement of the invention, the AgilentZORBAXSB-C18 column in step S3 has a specification of 250mm × 4.6mm and a particle size of 5 μm.

As a further improvement of the present invention, the chromatographic conditions in step S3: adopting an AgilentZORBAXSB-C18 column, wherein a mobile phase A is acetonitrile, a mobile phase B is 0.1wt% formic acid solution, gradient elution is adopted, the flow rate is 1ml/min, the column temperature is 30 ℃, and the detection wavelength is as follows: 300nm; the amount of sample was 10. Mu.l.

As a further improvement of the present invention, the gradient elution conditions in step S3 are: 5min,5% phase A; 5 → 20min,5 → 12% of phase A; 20 → 40min,12 → 20% phase A; 40 → 50min,20% phase A; 50 → 65min,20 → 90% phase A; 65 → 80min,90% of phase A.

The invention further protects the Juanbi decoction substance standard HPLC fingerprint obtained by the determination method.

As a further improvement of the invention, 11 common characteristic peaks are formed on the chromatogram, the common characteristic peaks are numbered from left to right, and the No. 6 chromatographic peak is ferulic acid.

The invention has the following beneficial effects: fingerprint spectrum of Juanbi decoction is determined by HPLC-UV method for 10 batches, and the fingerprint spectrum of Juanbi decoction is established with the chromatographic peak of ferulic acid as reference peak. Through analyzing 10 batches of Bi Tang fingerprint spectrums, 11 common peaks are formed; and comparing the spectra of the medicinal materials with the spectra of the samples to determine the attribution of the common peaks. The fingerprint spectrum measuring method based on Juanbi decoction substance has strong specificity, better repeatability and stability, simple method and better control of the internal quality of the product, so the fingerprint spectrum measuring based on Juanbi decoction substance is used as the quality control method of the product.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a model chart of the Juanbi decoction substance standard HPLC fingerprint chromatogram;

FIG. 2 is a superimposed view of HPLC fingerprint spectra of the material standard of Juanbi decoction of 10 batches;

FIG. 3 is an HPLC chart of a mixed control solution.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The apparatus used in the embodiment of the present invention: LC-2030C-3D high performance liquid chromatograph (Shimadzu corporation); ultrasonic cleaner (SG 250HE, shanghai guan ultrasonic instruments ltd); electronic balance (one hundred thousand, BT 125D), sydows scientific instruments (beijing) ltd).

The chemical reagents used in the embodiments of the present invention: chlorogenic acid (B20782, content 98%, shanghai-sourced leaf Biotechnology, inc.); gentiopicroside (190513, content 98%, institute for food and drug assay, china); ferulic acid (110773-201614, 99% content, institute for Chinese food and drug assay), liquiritin (111610-201607, 93.1% content, institute for Chinese food and drug assay), and Digitalis purpurea lactone I (MUST-20083105, 99.43% content, institute for Chengdu biology, china academy of sciences); acetonitrile (chromatographic grade, fisher corporation); methanol, formic acid (analytical grade, chemical reagents of national drug group, ltd.); ultrapure water.

The material standards of 10 batches of Juanbi decoction related in this example are provided by pharmaceutical factories of Guangxi Chinese medicine university, and raw materials used in experiments are purchased in pharmacies. The information on the drug substances of the samples is shown in Table 1.

TABLE 2 medicinal material information

Medicinal materials	Origin of birth	Source	Batch number
				Notopterygium root	Sichuan	Wanyaotang pharmaceutical Co Ltd in Nanning City	190401
Radix Angelicae Pubescentis	Sichuan	Wanyaotang pharmaceutical Co Ltd in Nanning City	180901
				Cortex Cinnamomi	Guangxi province	Guangxi Xianzhu traditional Chinese Medicine Technology Co.,Ltd.	20191001
Radix Gentianae Macrophyllae	Chongqing	Guangxi Xianzhu traditional Chinese Medicine Technology Co.,Ltd.	20190101
				Radix Angelicae sinensis	Gansu	Nanning Binyang Wanrun materia medica Co.,Ltd.	19110801
Rhizoma Ligustici Chuanxiong	Sichuan province	Guangxi Xianzhu traditional Chinese Medicine Technology Co.,Ltd.	20191001
				Licorice root (moxibustion)	Gansu	Guangxi Xianzhu traditional Chinese Medicine Technology Co.,Ltd.	20191201
Caulis Piperis Futokadsurae	Guangxi province	Guangxi Xianzhu traditional Chinese Medicine Technology Co.,Ltd.	20180501
				Ramulus Mori	Guangxi province	Guangxi Xianzhu traditional Chinese Medicine Technology Co.,Ltd.	20190601
Olibanum (Olibanum)	Guangdong (Chinese character of 'Guangdong')	Limited liability company of Guangxi Tongftang Chinese herbal pieces	170501
				Radix aucklandiae	Yunnan province	Guangxi Xianzhu traditional Chinese Medicine Technology Co.,Ltd.	20190401

Example 1

The embodiment provides a method for measuring a standard HPLC fingerprint spectrum of Juanbi decoction material, which comprises the following steps:

s1, preparation of a reference substance solution: precisely weighing appropriate amount of chlorogenic acid, gentiopicrin, ferulic acid, liquiritin and senkyunolide I, adding methanol for dissolving, and respectively making into mixed reference substance solutions with concentrations of 0.19mg/ml, 0.09-0.11mg/ml and 0.09 mg/ml;

s2, preparing a test solution: precisely weighing 2g of Juanbi decoction, placing in a volumetric flask, adding methanol to constant volume, ultrasonically extracting for 30min at power of 180W and frequency of 35kHz, cooling to room temperature, supplementing lost weight, shaking up, filtering, and filtering with 0.22 micron microporous membrane to obtain sample solution;

s3, chromatographic conditions: an AgilentZORBAXSB-C18 column (specification of 250mm × 4.6mm, particle size of 5 μm) is adopted, mobile phase A is acetonitrile, phase B is 0.05wt% formic acid solution, gradient elution is adopted, flow rate is 0.5ml/min, column temperature is 28 ℃, detection wavelength: 280nm; the sample amount is 5 mul;

the gradient elution conditions were: 5min,5% phase A; 5 → 20min,5 → 12% of phase A; 20 → 40min,12 → 20% phase A; 40 → 50min,20% phase A; 50 → 65min,20 → 90% phase A; 65 → 80min,90% phase A;

Example 2

s1, preparation of a reference substance solution: precisely weighing appropriate amount of chlorogenic acid, gentiopicrin, ferulic acid, liquiritin and senkyunolide I, dissolving with methanol, and respectively making into mixed reference solutions with concentrations of 0.21mg/ml, 0.11mg/ml and 0.11 mg/ml;

s2, preparation of a test solution: precisely weighing 2g of Juanbi decoction, placing in a volumetric flask, adding methanol to constant volume, ultrasonically extracting for 50min at 220W power and 45kHz frequency, cooling to room temperature, supplementing lost weight, shaking, filtering, and filtering with 0.22 μm microporous membrane to obtain sample solution;

s3, chromatographic conditions: an AgilentZORBAXSB-C18 column (specification is 250mm multiplied by 4.6mm, particle size is 5 mu m), acetonitrile is taken as a mobile phase A, 0.15wt% formic acid solution is taken as a phase B, gradient elution is adopted, flow rate is 1.5ml/min, column temperature is 32 ℃, detection wavelength: 320nm; the sample amount is 15 mul;

the gradient elution conditions were: 5min,5% phase A; 5 → 20min,5 → 12% phase A; 20 → 40min,12 → 20% phase A; 40 → 50min,20% phase A; 50 → 65min,20 → 90% phase A; 65 → 80min,90% phase A;

Example 3

The embodiment provides a method for measuring standard HPLC fingerprint of Juanbi decoction, which comprises the following steps:

s1, preparation of a reference substance solution: precisely weighing appropriate amount of chlorogenic acid, gentiopicrin, ferulic acid, liquiritin and senkyunolide I, dissolving with methanol, and respectively making into mixed reference solutions with concentrations of 0.197mg/ml, 0.1984mg/ml, 0.2057mg/ml, 0.0936 mg/ml and 0.1050 mg/ml;

s2, preparing a test solution: precisely weighing 2g of Juanbi decoction, placing in a volumetric flask, adding methanol to constant volume, ultrasonically extracting for 40min at 200W and 40kHz frequency, cooling to room temperature, supplementing lost weight, shaking, filtering, and filtering with 0.22 μm microporous membrane to obtain sample solution;

s3, chromatographic conditions: an AgilentZORBAXSB-C18 column (specification is 250mm multiplied by 4.6mm, particle size is 5 mu m), acetonitrile is used as a mobile phase A, 0.05-0.15wt% formic acid solution is used as a phase B, gradient elution is adopted, flow rate is 1ml/min, column temperature is 30 ℃, detection wavelength: 300nm; the sample amount is 10 mul;

the gradient elution conditions were: 5min,5% of phase A; 5 → 20min,5 → 12% of phase A; 20 → 40min,12 → 20% phase A; 40 → 50min,20% phase A; 50 → 65min,20 → 90% phase A; 65 → 80min,90% phase A;

Example 4 precision test

Taking 2g of Juanbi decoction material standard (batch number: 20190201), preparing a test solution according to the method of the step S2 in the embodiment 3, measuring according to chromatographic conditions, and continuously injecting samples for 6 times. The relative retention time of the other 11 common peaks except the ferulic acid is respectively recorded, and the experimental result shows that the RSD value of the relative retention time of the 11 common peaks is between 0.57 and 1.7 percent, and the RSD value of the relative peak area is between 0.8 and 1.9 percent, which indicates that the precision of the instrument is good and meets the related requirements and regulations of fingerprint spectrum determination.

Example 5 stability test

Sample solutions were prepared according to the method of step S2 in example 3, taking 2g of Juanbi decoction material basis (lot number: 20190201), and measured according to chromatographic conditions at 0, 2, 4, 8, 12, and 24h, respectively. And taking the retention time of the ferulic acid and the chromatographic peak area as references, and recording the retention time and the peak area of the common peak. The test result shows that the relative retention time RSD value of 11 common peaks is between 0.40 and 1.87 percent, and the relative peak area RSD is between 0.93 and 1.91 percent, which indicates that the sample is stable within 24h and meets the related requirements and regulations of fingerprint determination.

Example 6 repeatability test

Taking 6 parts of the same batch of Juanbi decoction material basis (batch numbers: 20190201, 20190202, 20190203, 20190204, 20190205 and 20190206), preparing a sample solution according to the method of the step S2 in the embodiment 3, measuring according to chromatographic conditions, and recording the retention time and the peak area of the common peak by taking the retention time and the peak area of the chromatographic peak as references. The test result shows that the relative retention time RSD value of 11 common peaks is between 0.64 and 1.98 percent, and the relative peak area RSD is between 0.82 and 1.88 percent, which indicates that the method has good repeatability and meets the related requirements and regulations of fingerprint determination.

Example 7 fingerprint

1. Determination of fingerprint

Taking 10 batches of Juanbi decoction material standard, preparing a sample solution according to the method of the step S2 in the embodiment 3, measuring according to chromatographic conditions, recording a chromatogram of a sample, and meeting related requirements and regulations of fingerprint spectrum measurement.

Based on 10 batches of HPLC chromatograms of Juanbi decoction material standard, establishing a common mode chromatogram of the Juanbi decoction material standard HPLC fingerprint chromatogram, analyzing by adopting a traditional Chinese medicine chromatogram fingerprint chromatogram similarity evaluation system (see figure 1), selecting the fingerprint of the Juanbi decoction material standard sample with the batch number of 20190201 as a reference chromatogram, generating the common mode chromatogram after multi-point correction and automatic matching, determining 11 common peaks, and establishing the common mode reference chromatogram (figure 2).

2. HPLC (high Performance liquid chromatography) spectrum determination and characteristic peak attribution analysis of reference substances

Precisely measuring and analyzing the mixed reference substance solution under chromatographic conditions, recording a chromatogram, comparing the chromatogram with common peaks in a sample fingerprint, and determining that the retention time of

characteristic peaks

3, 4, 7, 8 and 9 in the common fingerprint is consistent with that of chlorogenic acid, gentiopicrin, ferulic acid, liquiritin and senkyunolide I reference substances, so that the

peaks

3, 4, 7, 8 and 9 in the Juanbi soup quality reference spectrum are chlorogenic acid, gentiopicrin, ferulic acid, liquiritin, decursin and senkyunolide I. See fig. 3.

3. Analysis of common peaks

By analyzing 10 batches of sample chromatograms, selecting chromatographic peaks with good repeatability and obvious characteristics as common peaks, marking 11 common peaks in total, selecting No. 7 peak (ferulic acid) with relatively large peak area, intermediate retention time and good stability as a reference peak in the sample chromatogram, and respectively calculating the relative peak area and the relative retention time of the other 10 peaks except the ferulic acid. RSD of 10 samples with relative retention time is less than 0.77%, meeting the relevant regulations, and RSD of relative peak area is 23.87% minimum and 62.92% maximum, which may be related to the difference of original material production of the preparation or the difference generated in the preparation production process.

4. Similarity analysis

According to the evaluation of similarity of traditional Chinese medicine chromatogram fingerprints (2012.130723 edition), 10 batches of Juanbi soup substance-based fingerprints are processed and calculated in detection data, and the result shows that the similarity of 10 batches of Juanbi soup substance-based fingerprints is more than 0.9, the similarity is good, and the analysis requirements of the fingerprints are met. The results are shown in Table 2.

TABLE 210 Juanbi decoction material-based similarity evaluation results

Sample(s)	Degree of similarity	Sample(s)	Similarity of the two
				S1	0.954	S6	0.989
S2	0.995	S7	0.913
				S3	0.997	S8	0.962
S4	0.994	S9	0.986
				S5	0.971	S10	0.989

In conclusion, the fingerprint spectrum measuring method based on Juanbi decoction substance has strong specificity, better reproducibility and stability, simple method and better aim of controlling the internal quality of the product, so the fingerprint spectrum measuring based on Juanbi decoction substance is taken as the quality control method of the product.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for measuring a standard HPLC fingerprint spectrum of Juanbi decoction substances is characterized by comprising the following steps:

s1, preparation of a reference substance solution: precisely weighing appropriate amount of chlorogenic acid, gentiopicrin, ferulic acid, liquiritin and senkyunolide I, dissolving with methanol, and respectively making into mixed reference solution;

s2, preparation of a test solution: precisely weighing the standard of the Juanbi decoction, placing in a volumetric flask, adding methanol to constant volume, ultrasonically extracting, cooling to room temperature, complementing the lost weight, shaking up, and filtering to obtain a test solution;

s3, chromatographic conditions: adopting an AgilentZORBAXSB-C18 column, wherein the mobile phase A is acetonitrile, the mobile phase B is 0.05-0.15wt% formic acid solution, adopting gradient elution, the flow rate is 0.5-1.5ml/min, the column temperature is 28-32 ℃, and the detection wavelength is as follows: 280-320nm; the sample amount is 5-15 mul;

s4, determination: respectively and precisely sucking the reference solution and the test solution, injecting into a high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step S3.

2. The assay method according to claim 1, wherein the concentrations of chlorogenic acid, gentiopicroside, ferulic acid, liquiritin and senkyunolide I in the mixed control solution in step S1 are 0.19-0.21mg/ml, 0.09-0.11mg/ml and 0.09-0.11mg/ml.

3. The method according to claim 1, wherein the ultrasonic extraction time in step S2 is 30-50min, the power is 180-220W, and the frequency is 35-45kHz.

4. The method according to claim 3, wherein the ultrasonic extraction time in step S2 is 40min, the power is 200W, and the frequency is 40kHz.

5. The method of claim 1, wherein the AgilentZORBAXSB-C18 column at step S3 has a size of 250mm x 4.6mm and a particle size of 5 μm.

6. The method according to claim 1, wherein the chromatographic conditions in step S3 are: an AgilentZORBAXSB-C18 column is adopted, the mobile phase A is acetonitrile, the mobile phase B is 0.1wt% formic acid solution, gradient elution is adopted, the flow rate is 1ml/min, the column temperature is 30 ℃, and the detection wavelength is as follows: 300nm; the amount of sample was 10. Mu.l.

7. The method according to claim 1, wherein the gradient elution conditions in step S3 are: 5min,5% of phase A; 5 → 20min,5 → 12% phase A; 20 → 40min,12 → 20% phase A; 40 → 50min,20% phase A; 50 → 65min,20 → 90% phase A; 65 → 80min,90% of phase A.

8. A Juanbi decoction substance reference HPLC fingerprint obtained by the determination method according to any one of claims 1-7.

9. The substance-based HPLC fingerprint of Juanbi decoction according to claim 8, wherein 11 common characteristic peaks are formed on the chromatogram, and the number 6 chromatographic peak is ferulic acid, which are numbered from left to right.