WO2017148418A1

WO2017148418A1 - Method for determining component contents of chinese medicine composition

Info

Publication number: WO2017148418A1
Application number: PCT/CN2017/075456
Authority: WO
Inventors: 陈育鹏; 毕丹; 赵倩; 张水英; 叶玉廷; 任晋; 魏峰
Original assignee: 石家庄以岭药业股份有限公司
Priority date: 2016-03-03
Filing date: 2017-03-02
Publication date: 2017-09-08
Also published as: CN107149623A; CN107149623B

Abstract

Provided is a method for determining the component contents of a Chinese medicine composition. Raw materials for the Chinese medicine composition consist of forsythia, Japanese honeysuckle, indigowoad root, bitter almond, mentha-camphor, houttuynia cordata, rhubarb, patchouli, male fern rhizome, kirilow rhodiola root and rhizome, ephedra, licorice, and gypsum. The component content determination method uses a high-performance liquid chromatography technique to simultaneously determine the contents of seven components: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, 4,5-di-O-caffeoylquinic acid, quercitrin, and glycyrrhizic acid ammonium salt. A test sample is extracted by methanol or ethanol. The chromatographic conditions comprise using a methanol/acetonitrile/0.1-0.5% phosphoric acid aqueous solution as a mobile phase to perform a gradient elution, and using two detection wavelengths of 327 nm and 250 nm.

Description

Method for determining content of traditional Chinese medicine composition

Technical field

The invention relates to a method for determining the content of a traditional Chinese medicine composition, and belongs to the technical field of quality control methods for medicines.

Background technique

Traditional Chinese medicine is the most important material means for Chinese medicine to prevent and cure diseases. The modernization of traditional Chinese medicine that has been implemented since the "Ninth Five-Year Plan" in China is a major measure to promote the development of the Chinese medicine industry. The quality control and evaluation of traditional Chinese medicine is one of the key issues in the modernization of traditional Chinese medicine, and it is also a difficult point and hot spot in the research of traditional Chinese medicine. The purpose of quality control of traditional Chinese medicine is to ensure the effectiveness and safety of traditional Chinese medicine. The quality control of traditional Chinese medicine is to monitor the pharmacodynamic substances, toxic substances and their changing laws of traditional Chinese medicine. It is based on a series of quality standards to conduct quality inspection and control on every link in the production process of traditional Chinese medicine. The research on the techniques, methods and strategies of quality evaluation of traditional Chinese medicine is the basis for formulating scientific, reasonable and advanced quality standards. Liquid chromatography has a high degree of separation, which can separate complex chemical components to form different chromatograms. The height and peak area of these peaks represent various chemical components and their contents. Its content can effectively reflect the quality of the product and its stability. At present, the content determination method is mainly composed of a single component, and there is rarely one method for simultaneously determining the content determination method of a plurality of active ingredients.

The invention claims a method for determining the content of a traditional Chinese medicine composition, which comprises the following herbs: forsythia, honeysuckle, radix isatidis, bitter almond, menthol, houttuynia, rhubarb, patchouli, and cotton horse , Rhodiola, Ephedra, Licorice, Gypsum, the content determination method, simultaneous determination of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl The content of seven components of linic acid, quercetin and glycyrrhizic acid can effectively save energy and reduce the analysis cost, and the technical content is not disclosed in the prior art.

Summary of the invention

The invention provides a method for determining the content of a traditional Chinese medicine composition. The method can simultaneously determine various active ingredients in the traditional Chinese medicine composition.

A method for determining the content of a traditional Chinese medicine composition, the raw material medicine of the traditional Chinese medicine composition comprises: forsythia, ephedra, rhubarb, houttuynia, honeysuckle, radix isatidis, patchouli, Mianma Guanzhong, Rhodiola, menthol, Bitter almond, licorice, gypsum, characterized in that the content determination method comprises the following steps:

Preparation of the test solution: taking the traditional Chinese medicine composition, and extracting with an organic solvent to obtain a test solution;

Preparation of reference solution: take appropriate amount of chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate , dissolved in an organic solvent to obtain a reference solution;

Chromatographic conditions: using a methanol-acetonitrile-phosphoric acid aqueous solution ternary solvent system as a mobile phase, gradient elution, the concentration of the aqueous phosphoric acid solution is 0.1% to 0.5%;

Determination method: separately draw the reference solution and the test solution, inject into the liquid chromatograph, and measure, that is, obtain.

Preferably, the organic solvent comprises 50% to 100% methanol solution, 50% to 100% ethanol solution.

Preferably, the gradient elution procedure is:

Preferably, the detection wavelengths are 327 nm and 250 nm, respectively; quercetin and glycyrrhizic acid are detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5- are detected at 327 nm. Di-O-caffeoylquinic acid.

Further preferably, the present invention provides a method for determining the content of a traditional Chinese medicine composition, which is prepared from the following raw materials by weight: Forsythia 200-300, Ephedra 60-100, Rhubarb 40-60, Houttuynia cordata 200-300, honeysuckle 200-300, Banlangen 200-300, patchouli 60-100, cotton horse Guanzhong 200-300, Rhodiola 60-100, menthol 5-9, bitter almond 60-100, licorice 60-100, gypsum 200-300, the content determination method is as follows:

Preparation of the test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 25mL of ethanol or methanol, extract it by ultrasonic or reflux for 30 minutes, filter, obtain the filtrate, and evaporate. The residue is dissolved in ethanol or methanol and made up to 25 mL, which is obtained;

Preparation of reference solution: taking fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate reference substance Appropriate amount, accurately weighed, add 50% methanol to make 34.4μg of new chlorogenic acid per 1mL, 76.7μg chlorogenic acid, 34.2μg cryptochlorogenic acid, 56.2μg of allosperglycin A, 14.9μg 4,5-two a mixed solution of -O-caffeoylquinic acid, 11.2 μg of quercetin and 42.8 μg of ammonium glycyrrhizinate, wherein, the weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207;

Chromatographic conditions: methanol-acetonitrile-0.1% phosphoric acid solution as mobile phase, gradient elution procedure is:

The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; the column is a C ₁₈ column;

Determination method: respectively, accurately draw 2 μL of the reference solution and the test solution, inject into the liquid chromatograph, measure and record the chromatogram, that is, obtain.

The preferred pharmaceutical ingredients of the traditional Chinese medicine composition of the present invention are: Forsythia 200, Honeysuckle 300, Banlangen 200, Rhubarb 40, Patchouli 60, Mianma Guanzhong 300, Rhodiola 100, Menthol 9, Ephedra 60, bitter almond 100, houttuynia cordica 200, licorice 100, gypsum 200.

The preferred parts of the traditional Chinese medicine composition of the present invention are: Forsythia 300, Honeysuckle 200, Banlangen 300, Rhubarb 60, Patchouli 100, Mianma Guanzhong 200, Rhodiola 60, Menthol 5, Ephedra 100, bitter almond 60, houttuyniasis 300, licorice 60, gypsum 300.

The preferred pharmaceutical ingredients of the traditional Chinese medicine composition of the present invention are: Forsythia 278, Honeysuckle 294, Radix Isatidis 285, Rhubarb 55, Patchouli 95, Mianma Guanzhong 290, Rhodiola 87, Menthol 8.5, Ephedra 88, bitter almond 80, houttuynia 284, licorice 95, gypsum 277.

The preferred pharmaceutical ingredients of the traditional Chinese medicine composition of the present invention are: Forsythia 255, Honeysuckle 255, Radix Isatidis 255, Rhubarb 51, Patchouli 85, Mian Ma Guan 255, Rhodiola 85, Menthol 7.5, Ephedra 85 , bitter almond 85, houttuynia 255, licorice 85, gypsum 255.

The preparation method of the traditional Chinese medicine composition preparation of the invention is:

(1) Weigh Chinese medicines according to the weight ratio of raw materials, net selection, and break as appropriate;

(2) Patchouli scented, add 10 times the amount of water to extract volatile oil, oil extraction time 8 hours, collect volatile oil, spare; after the extract is filtered, the residue is discarded, the filtrate is reserved;

(3) Forsythia, Ephedra, Houttuynia cordata, Rhubarb, extracted with 12 times 70% ethanol for 3 times, each time for 2.5 hours, the extract is combined with filtration, ethanol is recovered, and the filtrate is reserved;

(4) Honeysuckle, gypsum, Banlangen, Mianma Guanzhong, Licorice, Rhodiola, add 12 times the amount of water to boil, add bitter almonds, decoct 2 times, each time for 1 hour, extract and filter, the result The filtrate is combined with the filtrate of step (2) patchouli oil extraction, and concentrated to obtain a clear paste having a relative density of 1.10-15.15 at 60 ° C, added with ethanol, adjusted to an alcohol concentration of 70%, refrigerated, filtered, and recovered. Ethanol to no alcohol, get a clear paste;

(5) Combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), and concentrating to obtain a clear paste having a relative density of 1.15 - 1.20 at 60 ° C, and drying to obtain a dry paste powder, which is used;

(6) adding the dry paste powder obtained in the step (5) to a suitable pharmaceutically acceptable auxiliary material for granulation;

(7) menthol, the volatile oil obtained in the step (2) is dissolved in ethanol, sprayed into the granules obtained in the step (6), sealed, mixed, tableted or encapsulated, or Bagging.

The content determination method according to the present invention is preferably:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 50mL ethanol, 25mL, ultrasonic extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter, The filtrate was obtained, and the filter paper was added together with the residue into a stoppered conical flask, and 25 mL of 50% ethanol was added thereto, sonicated for 30 minutes, filtered, and the dregs were washed 4 times with 50% ethanol, 5 mL each time, and the filtrate and washing were combined twice. The liquid is evaporated to dryness, and the residue is dissolved in 50% methanol and made up to 25 mL, which is obtained;

The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; column: ACQUITY

HSS T3 (1.8 μm, 2.1 × 100 mm); column temperature is 35 ° C;

The content determination method according to the present invention is also preferably:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 50% methanol 25mL, ultrasonic extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter, The filtrate was obtained, and the filter paper was added together with the residue into a stoppered conical flask, and then 50 mL of 50% methanol was added thereto, sonicated for 30 minutes, filtered, and the dregs were washed 4 times with 50% methanol, 5 mL each time, and the filtrate and washing were combined twice. The liquid is evaporated to dryness, and the residue is dissolved in 50% methanol and made up to 25 mL, which is obtained;

The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; column is ACQUITY

CSH ^TM C ₁₈ ;

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 25mL of methanol, reflux extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter, and obtain filtrate Add the filter paper together with the residue into a stoppered conical flask, add 25mL of methanol, sonicate for 30 minutes, filter, wash the dregs with methanol 4 times, each time 5mL, combine the filtrate and washing solution twice, evaporate and dry, residue Dissolve with 50% methanol and dilute to 25mL, that is;

BEHC ₁₈ ;

The content determination method of the present invention is also preferably:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 25mL of ethanol, ultrasonic extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter, and obtain filtrate Add the filter paper together with the residue into the stoppered conical flask, add 25mL of ethanol, sonicate for 30 minutes, filter, wash the dregs with ethanol 4 times, each time 5mL, combine the filtrate and washing solution twice, evaporate and dry, residue Dissolve with 50% methanol and dilute to 25mL, that is;

BEHC ₁₈ ;

Using the sample prepared in Example 1, the method for determining the content of the present invention evaluates the feasibility of the method from various aspects, and the evaluation method is as follows:

1. Instruments, reagents and reagents

1.1 Instrument

US Waters ACQUITY UPLC H-CLASS Ultra High Performance Liquid Chromatograph (TUV Detector, Empower 3.0 Chromatography Workstation), Circulating Multipurpose Vacuum Pump (Zhengzhou Changcheng Branch Industry and Trade Co., Ltd.), KQ250DB Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) ), Switzerland METTLER TOLEDOAL204 electronic analytical balance, Switzerland METTLER TOLEDOAB135-S electronic analytical balance, Shanghai Jingke JA2603B electronic balance.

1.2 reagent

Ethanol (analytical grade, Tianjin Guangfu Technology Development Co., Ltd.), phosphoric acid (analytical grade, Tianjin Guangfu Technology Development Co., Ltd.), acetonitrile (chromatographically pure, Fisher Company, USA), methanol (chromatographically pure, Fisher Company, USA), new green Acid (Chengdu Puruifa Technology Development Co., Ltd., batch number: 13112712, purity ≥98%), chlorogenic acid (China Food and Drug Testing Institute, batch number: 110753-201314, purity, 96.6%), cryptochlorogenic acid (Shanghai Source Leaf Biotechnology Co., Ltd., batch number: ZF0226BA14, purity ≥98%), isophoric acid ester A (Shanghai Yuanye Biotechnology Co., Ltd., batch number: YA0817HB14, purity ≥98%), 4,5-di-O- Caffeoylquinic acid (China Food and Drug Control Institute, batch number: 111894-201102, purity, 94.1%), saponin (HWIANALYTIK GMBH pharma solutions, batch number: HWI01112, purity, 91.7%), ammonium glycinate (Council of Europe) - EDQM, batch number: 1.1, purity ≥ 98%).

1.3 reagent: the traditional Chinese medicine composition of the invention

2. Chromatographic conditions

2.1 Selection of detection wavelength

According to the UV spectrum of the reference solution of the component to be tested, the maximum absorption peak at 327 nm and 250 nm was selected as the detection wavelength, and the new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4 were detected at 327 nm. , 5-di-O-caffeoylquinic acid, quercetin and glycyrrhizic acid were detected at 250 nm to minimize interference from other components.

2.2 Choice of mobile phase

The different ratios of acetonitrile-methanol-0.1% phosphoric acid mobile phase system were investigated. The results showed that the chromatographic peaks eluted by the gradient elution in the following table had good resolution and the baseline was stable. There was no obvious impurity interference around the chromatographic peaks to be tested.

Gradient elution table

2.3 Determination of chromatographic conditions

A methanol-acetonitrile-0.1% phosphoric acid solution was used as a mobile phase, and a gradient elution was carried out according to the above table. The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; column: ACQUITY

HSS T3 (1.8 μm, 2.1 x 100 mm); column temperature was 35 °C.

3.1 Investigation of extraction solvent

Take the appropriate amount of the traditional Chinese medicine composition of the present invention, and grind it, take 8 parts, 0.5g each, accurately weighed, place it in a conical flask, and add 25mL of methanol, ethanol, 50% methanol and 50% ethanol respectively. Ultrasonic treatment (power 500W, frequency 40kHz) for 30 minutes, filtered, the filtrate was obtained, the filter paper was added together with the residue into a stoppered conical flask, and then added with 25 mL of methanol, ethanol, 50% methanol and 50% ethanol, respectively, and sonicated for 30 minutes. The mixture was filtered, and the dregs were washed 4 times with 5 ml each time, and the washing liquid and the filtrate were combined, evaporated to dryness, and the residue was dissolved in 50% methanol and made up to 25 ml, as a test solution.

2 μL of the test solution was separately taken and injected into a liquid chromatograph to measure the peak area of the component to be tested in the test sample. The results are shown in Table 1.

Table 1 Experimental results of different extraction solvents

The experimental results show that when 50% ethanol is used as the extraction solvent, the extraction rate of the components to be tested is the highest.

3.2 Investigation of extraction methods

Take the appropriate amount of the traditional Chinese medicine composition of the present invention, and grind it, take 4 parts, 0.5g each, accurately weighed, place it in a conical flask, add 50mL of 50% ethanol, and extract 2 times each with reflux for 2 hours and ultrasound. The extract was extracted by two different extraction methods for 30 minutes (2 times), the extract was filtered, the dregs were washed 4 times with 50% ethanol, 5 ml each time, the washing liquid was combined with the filtrate, evaporated to dryness, and the residue was dissolved in 50% methanol. And to a volume of 25ml, as a test solution. 2 μL of each test solution was accurately aspirated and injected into a liquid chromatograph to determine the peak area of the component to be tested in the test solution. The results are shown in Table 2.

Table 2 Experimental results of different extraction methods

The experimental results show that the reflux extraction is slightly higher than the ultrasonic extraction peak area, and can be used as the extraction method of the traditional Chinese medicine composition of the present invention.

3.3 Investigation of the number of extractions

The ultrasonic extraction was investigated once, twice and three times, and the extraction effects of different extraction times were compared. The results are shown in Table 3 below:

Table 3 Investigation of different extraction times

The experimental results show that the extraction efficiency of the components to be tested is the highest when extracted three times, but the extraction efficiency is only 1-2% higher than that of the extraction three times, which indicates that the results of extraction and extraction are not much different.

3.4 Determination of the preparation method of the test solution

Take the proper amount of the traditional Chinese medicine composition of the present invention, grind it, take 0.5g, accurately weigh it, place it in a conical flask, add 25mL of 50% ethanol, sonicate (power 500W, frequency 40kHz) for 30 minutes, filter, and obtain the filtrate. Add the filter paper together with the residue into a stoppered conical flask, add 25mL of 50% ethanol, sonicate for 30 minutes, filter, wash the dregs with 50% ethanol 4 times, each time 5mL, combine the filtrate and washing solution twice, steam Dry, the residue was dissolved in 50% methanol and made up to 25 mL.

Preparation of reference solution

Take the appropriate amount of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate, accurately weighed, Add 50% methanol to make 34.4μg of new chlorogenic acid per 1mL, 76.7μg chlorogenic acid, 34.2μg cryptochlorogenic acid, 56.2μg of allosperglycin A, 14.9μg 4,5-di-O-caffeoyl A mixed solution of n-acid, 11.2 μg of quercetin and 42.8 μg of ammonium glycyrrhizinate was obtained (weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207).

Linear relationship

Weigh accurately 15.56mg of new chlorogenic acid and 15.66mg of cryptochlorogenic acid in a 10ml volumetric flask, dissolve it with 50% methanol and dilute to the scale as the reference mother liquor 1; accurately weigh 15.35mg chlorogenic acid and 11.24mg respectively. The isophoraside A was placed in a 10 ml volumetric flask, dissolved in 50% methanol and diluted to the mark as the reference mother liquor 2; respectively, 15.88 mg of 4,5-di-O-caffeoylquinic acid and 12.24 mg of cesium were accurately weighed respectively. The lycopene reference substance was placed in a 10 ml volumetric flask and dissolved in 50% methanol and diluted to the scale as the reference mother liquid 3, respectively. 10.69 mg of ammonium glycyrrhizinate reference substance was accurately weighed and placed in a 10 ml volumetric flask and dissolved in 50% methanol. Dilute to the scale as the reference mother liquor 4, and then accurately extract 1ml, 3ml, 1ml, 2ml from the

reference mother liquor

1, 2, 3, 4 into a 10ml volumetric flask, and dilute to the scale with 50% methanol as a reference. The stock solution is then accurately extracted from the stock solution by 0.1, 0.2, 0.5, 1, 2, 4 ml in a 10 ml volumetric flask, and the volume is adjusted to the mark with 50% methanol to obtain a series of reference solutions of different mass concentrations. .

Precision extraction of fresh chlorogenic acid 1.56μg/mL, 3.12μg/mL, 7.78μg/mL, 15.56μg/mL, 31.12μg/mL and 62.24μg/mL, containing chlorogenic acid 4.45μg/mL, 8.90μg/mL 22.24 μg/mL, 44.48 μg/mL, 88.97 μg/mL, and 177.94 μg/mL, containing cryptochlorogenic acid 1.57 μg/mL, 3.13 μg/mL, 7.83 μg/mL, 15.66 μg/mL, 31.32 μg/mL 62.64 μg/mL, containing forathiaside A3.37 μg/mL, 6.74 μg/mL, 16.86 μg/mL, 33.72 μg/mL, 67.44 μg/mL, 134.88 μg/mL, containing 4,5-di- O-caffeoyl quinic acid 1.49 μg/mL, 2.99 μg/mL, 7.47 μg/mL, 14.94 μg/mL, 29.89 μg/mL, 59.77 μg/mL, containing quercetin 1.12 μg/mL, 2.24 μg/mL 5.61 μg/mL, 11.22 μg/mL, 22.45 μg/mL, 44.90 μg/mL, containing glycyrrhizic acid 2.09 μg/mL, 4.19 μg/mL, 10.47 μg/mL, 20.95 μg/mL, 41.89 μg/mL, 83.79 2 μL of each of the μg/mL series of reference solutions were injected into the ultra-high performance liquid chromatograph, and determined according to the proposed chromatographic conditions. The mass concentration of the reference substance was plotted on the abscissa and the peak area was plotted on the ordinate. The standard curve was drawn to obtain the new chlorogenic acid. Regression equation: y=20628x-1699.7 (R ² =0.9999), the results show that the new chlorogenic acid is in the range of 1.56μg/mL~6 The linearity was good in the range of 2.24μg/mL; the regression equation of chlorogenic acid: y=19778x+8305.9 (R ² =0.9999), the results showed that the chlorogenic acid was linear in the range of 4.45μg/mL～177.94μg/mL; The acid regression equation: y=14319x-933.94 (R ² =0.9999), the results showed that the cryptochlorogenic acid was linear in the range of 1.57μg/mL～62.64μg/mL; the regression equation of the forsythin A: y=7951.8x -2304.5 (R ² =0.9999), the results showed that the forsythiaside A was linearly good in the range of 3.37 μg/mL to 134.88 μg/mL; the regression equation of 4,5-di-O-caffeoylquinic acid was: y =20148x-2422.8 (R ² =0.9999), the results showed that 4,5-di-O-caffeoylquinic acid was linear in the range of 1.49 μg / mL ~ 59.77 μg / mL; ecdysin regression equation: y = 13288x -3121.6 (R ² =0.9999), the results showed that quercetin was linearly good in the range of 1.12 μg/mL to 44.90 μg/mL; the regression equation of glycyrrhizic acid was: y=2731.3x+810.9 (R ² =0.9999), the results showed The glycyrrhizic acid has a good linearity in the range of 2.09 μg/mL to 83.79 μg/mL. The experimental results are shown in Table 4-7.

Table 4 Linear relationship between new chlorogenic acid and chlorogenic acid reference solution

Table 5 Linear relationship between cryptochlorogenic acid and isoprehtilin A reference solution

Table 6 Linear relationship between 4,5-di-O-caffeoylquinic acid and quercetin reference solution

Table 7 Linear relationship between glycyrrhizic acid reference solution

5. Determination of detection limits and limit of quantitation

Dilute the mixed reference solution of the component to be tested into a series of solutions, perform UPLC detection, record the ratio of signal intensity to noise intensity, and use the concentration of signal intensity and noise intensity equal to 3 as the detection limit, and the signal intensity and The concentration at which the noise intensity ratio is equal to 10 is taken as the limit of quantification. The detection limits of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, quercetin, 4,5-di-O-caffeoylquinic acid and glycyrrhizic acid were determined to be 0.051 μg/ mL, 0.049 μg/mL, 0.052 μg/mL, 0.111 μg/mL, 0.037 μg/mL, 0.049 μg/mL, and 2.095 μg/mL; the limits of quantification were 0.156 μg/mL, 0.147 μg/mL, and 0.157 μg/mL, respectively. 0.337 μg/mL, 0.112 μg/mL, 0.149 μg/mL, and 6.200 μg/mL.

Precision inspection

Take the reference solution (concentration: new chlorogenic acid 31.12μg/mL; chlorogenic acid 88.97μg/mL; cryptogenic chlorogenic acid 31.32μg/mL; avermectin A67.44μg/mL; 4,5-di- O-caffeoylquinic acid 29.89μg/mL; quercetin 22.45μg/mL; glycyrrhizic acid 41.89μg/mL) was continuously injected 6 times, the peak area was recorded, and the relative standard deviation (RSD) of each chromatographic peak area was calculated. To examine the precision of the instrument, the results are shown in Table 8.

Table 8 Precision inspection

The RSD of the peak area of the component to be tested is less than 2%, indicating that the precision of the instrument is good.

Repetitive investigation

Taking the same batch of the traditional Chinese medicine composition of the present invention, the samples were sampled according to the sample amount of "preparation of the test solution" at 80%, 100%, and 120%, and each sample was prepared in three portions, and determined according to law. The RSD is calculated based on the content of each component to be tested, and the results are shown in Table 9.

Table 9 Repeatability test results

It can be seen from the measurement results that the RSD of the content of the component to be tested is less than 3%, indicating that the method has good repeatability.

Stability investigation

The test solution was accurately taken and injected at room temperature for 0, 2, 4, 6, 8 and 12 hours, respectively. The peak area of the component to be tested was recorded, and the RSD of the peak area was calculated. The stability was investigated. The measurement results are shown in Table 10.

Table 10 Stability test results

It can be seen from the measurement results that the RSD of the peak area of the component to be tested is within 3%, indicating that the test solution is stable within 12 hours.

Sample recovery rate

According to the sampling amount of 0.5g of one-half, that is, 0.25g, weigh the product, accurately weighed, paralleled 9 parts, divided into 3 groups, respectively added low, medium and high three kinds of mass concentration of the reference substance to be tested The solution is prepared according to the "preparation method of the test solution", and is determined according to law, and the recovery rate test is investigated. The results are shown in Table 11-17.

Table 11 Investigation on the recovery rate of new chlorogenic acid

From the results of Table 11, it was found that the average recovery of new chlorogenic acid was 99.44%, and the RSD was 2.71%, which was in compliance with the requirements.

Table 12 Investigation on recovery rate of chlorogenic acid

From the results of Table 12, it was found that the average recovery of chlorogenic acid was 99.82%, and the RSD was 2.67%, which was in compliance with the requirements.

Table 13 Investigation on the recovery rate of cryptochloric acid

From the measurement results in Table 13, it was found that the average sample recovery rate of cryptochlorogenic acid was 97.61%, and the RSD was 1.64%, which was in compliance with the requirements.

Table 14 Investigation on the recovery rate of isophoric acid ester A

From the results of the measurement in Table 14, it was found that the average sample recovery rate of the forsythiaside A was 97.17%, and the RSD was 1.63%, which was in compliance with the requirements.

Table 15 Investigation of the recovery rate of quercetin

From the results of Table 15, it was found that the average recovery of quercetin was 99.19%, and the RSD was 2.89%, which was in compliance with the requirements.

Table 16 Investigation of the recovery rate of 4,5-di-O-caffeoylquinic acid

From the results of Table 16, it was found that the average recovery of 4,5-di-O-caffeoylquinic acid was 100.46%, and the RSD was 1.92%, which was in compliance with the requirements.

Table 17 Investigation on the recovery rate of glycyrrhizic acid

From the results of Table 17, it was found that the average recovery of glycyrrhizic acid was 100.32%, and the RSD was 0.97%, which was in compliance with the requirements.

System durability test

10.1 Effect of flow rate change on determination of multi-component content

The test solution was taken, and other chromatographic conditions were fixed, and the flow rates were 0.25 mL/min, 0.3 mL/min, and 0.35 mL/min, respectively, and the contents of the components to be tested were calculated according to the reference materials of the respective components. The effect of the change in flow rate on the measurement results was compared. The results are shown in Table 18.

Table 18 Effect of flow rate changes on the content of the components to be tested

From the above results, the RSD of cryptochlorogenic acid, quercetin and glycyrrhizic acid was more than 3% after the flow rate was changed, indicating that the flow velocity was slightly changed, and the influence on the three components in the sample was large.

10.2 Effect of wavelength change on the determination of the content of the component to be tested

The test solution was taken and other chromatographic conditions were fixed and measured at wavelengths of 322, 245 nm, 327, 250 nm, and 332, 255 nm, respectively, and the reference substance of each component to be tested was used as a control to calculate the content of the component to be tested, and to compare wavelength changes. The effect on the measurement results. The results are shown in Table 19.

Table 19 Effect of wavelength change on the content of the component to be tested

From the above results, it can be seen that after the wavelength change, the RSD value of the measurement results of cryptochlorogenic acid, quercetin and 4,5-di-Ocaffeoylquinic acid is more than 3%, indicating a slight change in wavelength, which is in the sample. The results of the determination of the three components have a great influence.

10.3 Effect of column temperature change on the determination of the content of the components to be tested

Take the test solution, fix other chromatographic conditions, measure at column temperature 30 °C, 35 °C and 40 °C, respectively, and compare the reference components of each component to be tested, calculate the content of the component to be tested, and compare the column temperature changes. The effect on the measurement results.

Table 20 Effect of column temperature change on the content of components to be tested

It can be seen from the above results that after the change of column temperature, the RSD values of chlorogenic acid, 4,5-di-O-caffeoylquinic acid and glycyrrhizic acid content are all greater than 3%, indicating that the column temperature is slightly changed to the sample. The content determination results of these three components have a great influence.

10.4 Effect of different chromatographic columns on the determination of the components to be tested

Take the test solution, fix other chromatographic conditions, use ACQUITY

CSH ^TM C ₁₈ (1.7μm, 2.1×100mm), ACQUITY

BEHC ₁₈ (1.7μm, 2.1×100mm), ACQUITY

BEH shieldRP 18 (1.7μm, 2.1×100mm) and ACQUITY

Four different columns, such as HSS T3 (1.8μm, 2.1×100mm), were tested to compare the effects of different chromatographic columns on the peak shape and resolution of the components to be tested. The results show that the content of the four components of the traditional Chinese medicine composition of the present invention can be used. When the HSS T3 column is used, the peak shape and the resolution of the component to be tested are the best.

Intermediate precision

According to the determined content determination method, the content of the same batch of the components of the traditional Chinese medicine composition of the present invention is determined by different analysts at different times and using two different UPLC instruments, and each test is operated in parallel for 3 times. Test the sample and examine the intermediate precision. The results are shown in Table 21-25.

Table 21 Analyst A uses UPLC (1) to determine the content of the component to be tested

Table 22 Analyst B uses UPLC (1) to determine the content of the component to be tested

Table 23 Analyst A uses UPLC (2) to determine the content of the component to be tested

Table 24 Analyst B uses UPLC (2) to determine the content of the component to be tested

Table 25 Intermediate precision inspection results

It can be seen from the above test results that the RSD values of the components of the traditional Chinese medicine composition of the present invention are less than 5% by different people at different times and different instruments, indicating that the precision of the method is good.

Determination of seven components in three batches of traditional Chinese medicine composition of the invention

The content of the components to be tested of the three batches of the traditional Chinese medicine composition of the present invention was measured by the determined content determination method, and the results are shown in Table 26.

Table 26 Determination of the content of the components to be tested in the capsule intermediate of the 3 batches of traditional Chinese medicine composition

The invention establishes a method for determining the content of seven components in the four herbs of the traditional Chinese medicine composition of the invention, and the methodological research results show that the method has the active ingredients in the four herbs of forsythia, honeysuckle, houttuynia and licorice. Good accuracy, precision, stability and repeatability can effectively control the quality and safety of the traditional Chinese medicine composition.

DRAWINGS

Figure 1: Chromatogram of the reference at 327nm, where 1 is neochlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is isopenic acid A, and 5 is 4,5-di-O-coffee. Acyl quinic acid

Figure 2: Chromatogram of the test solution at 327 nm

Figure 3: Chromatogram of the reference at 250 nm, where 6 is quercetin and 7 is glycyrrhizic acid

Figure 4: Chromatogram of the test solution at 250 nm

Figure 5: Chromatogram of Example 1: A is a chromatogram of the reference at 327 nm, where 1 is neochlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is isopenic acid A, 5 is 4,5-di-O-caffeoylquinic acid; B picture is the chromatogram of the test sample at 327nm; C picture is the chromatogram of the reference substance at 250nm, of which 6 is quercetin, 7 is glycyrrhizic acid; Chromatogram of the test sample at 250 nm.

Figure 6: Chromatogram of Example 2: Figure A is a chromatogram of the reference substance at 327 nm, where 1 is neochlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is isopenic acid A, 5 is 4,5-di-O-caffeoylquinic acid; B picture is the chromatogram of the test sample at 327nm; C picture is the chromatogram of the reference substance at 250nm, of which 6 is quercetin, 7 is glycyrrhizic acid; Chromatogram of the test sample at 250 nm.

Figure 7: Chromatogram of Example 3: Figure A is a chromatogram of the reference substance at 327 nm, where 1 is neochlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is isopenic acid A, 5 is 4,5-di-O-caffeoylquinic acid; B picture is the chromatogram of the test sample at 327nm; C picture is the chromatogram of the reference substance at 250nm, of which 6 is quercetin, 7 is glycyrrhizic acid; Chromatogram of the test sample at 250 nm.

Figure 8: Chromatogram of Example 4: Figure A is a chromatogram of the reference substance at 327 nm, where 1 is neochlorogenic acid, 2 is chlorogenic acid, 3 is cryptochlorogenic acid, 4 is isopenic acid A, 5 is 4,5-di-O-caffeoylquinic acid; B picture is the chromatogram of the test sample at 327nm; C picture is the chromatogram of the reference substance at 250nm, of which 6 is quercetin, 7 is glycyrrhizic acid; Chromatogram of the test sample at 250 nm.

detailed description

Example 1

Weighed according to the ratio: Forsythia 200g, Honeysuckle 300g, Radix Isatidis 200g, Rhubarb 40g, Patchouli 60g, Mianma Guanzhong 300g, Rhodiola 100g, Menthol 9g, Ephedra 60g, Bitter Almond 100g, Houttuynia 200g, Licorice 100g, gypsum 200g, extracted according to the following process:

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed, and capsules are obtained.

Method of determination:

HSS T3 (1.8 μm, 2.1 × 100 mm); column temperature is 35 ° C;

Conclusion: The results are satisfactory and can be used to control the quality of the traditional Chinese medicine composition.

Example 2

Weighed in proportion: Forsythia 300g, Honeysuckle 200g, Radix Isatidis 300g, Rhubarb 60g, Patchouli 100g, Mianma Guanzhong 200g, Rhodiola 60g, Menthol 5g, Ephedra 100g, Bitter Almond 60g, Houttuynia 300g, Glycyrrhiza 60g, gypsum 300g, extracted according to the following process:

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed and tableted.

Method of determination:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 50% methanol 25mL, ultrasonic extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter, obtain the filtrate, add the filter paper together with the residue into the conical flask, add 50mL methanol 25mL, sonicate for 30 minutes, filter, wash the dregs with 50% methanol 4 times 5mL each time, the filtrate and washing solution were combined twice, evaporated to dryness, and the residue was dissolved in 50% methanol and made up to 25 mL, which was obtained;

CSH ^TM C ₁₈ ;

Conclusion: Satisfactory results can be used to control the quality of the Chinese herbal composition.

Example 3

The raw material formula is: Forsythia 278g, Honeysuckle 294g, Radix Isatidis 285g, Rhubarb 55g, Patchouli 95g, Mianma Guanzhong 290g, Rhodiola 87g, Menthol 8.5g, Ephedra 88g, Bitter Almond 80g, Houttuynia 284g , licorice 95g, gypsum 277g, according to the following process:

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed, and the bag is obtained.

Method of determination:

BEH C ₁₈ ;

Results: Satisfactory results can be used to control the quality of the Chinese herbal composition.

Example 4

The raw material formula is: scaled: 200g of Forsythia, 300g of honeysuckle, 200g of Radix, 60g of rhubarb, 60g of patchouli, 300g of Mianmaguan, 60g of Rhodiola, 9g of menthol, 60g of ephedra, 100g of bitter almond, Houttuynia cordata 200g, licorice 100g, gypsum 200g, extracted according to the following process:

Method of determination:

Preparation of reference solution: taking fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate reference substance Appropriate amount, accurately weighed, add 50% methanol to make 34.4μg of new chlorogenic acid per 1mL, 76.7μg chlorogenic acid, 34.2μg cryptochlorogenic acid, 56.2μg of allosperglycin A, 14.9μg 4,5-two a mixed solution of -O-caffeoylquinic acid, 11.2 μg of quercetin and 42.8 μg of ammonium glycinate, wherein glycyrrhizic acid Weight = weight of ammonium glycyrrhizinate / 1.0207;

BEH shield RP 18;

Claims

A method for determining the content of a traditional Chinese medicine composition, the raw material medicine of the traditional Chinese medicine composition comprises: forsythia, ephedra, rhubarb, houttuynia, honeysuckle, radix isatidis, patchouli, Mianma Guanzhong, Rhodiola, menthol, Bitter almond, licorice, gypsum, characterized in that the content determination method comprises the following steps:

Preparation of the test solution: taking the traditional Chinese medicine composition, and extracting with an organic solvent to obtain a test solution;

Preparation of reference solution: take appropriate amount of chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate , dissolved in an organic solvent to obtain a reference solution;

Chromatographic conditions: using a methanol-acetonitrile-phosphoric acid aqueous solution ternary solvent system as a mobile phase, gradient elution, the concentration of the aqueous phosphoric acid solution is 0.1% to 0.5%;

Determination method: separately draw the reference solution and the test solution, inject into the liquid chromatograph, and measure, that is, obtain.
The content measuring method according to claim 1, wherein the organic solvent comprises 50% to 100% of a methanol solution and 50% to 100% of an ethanol solution.
The method of determining content according to claim 1, wherein said gradient elution procedure is:
The method for determining content according to claim 1, wherein the detection wavelengths are 327 nm and 250 nm, respectively; quercetin and glycyrrhizic acid are detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and different colors are detected at 327 nm. Forsythiaside A and 4,5-di-O-caffeoylquinic acid.
The content determination method according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials by weight: Forsythia 200-300, Ephedra 60-100, Rhubarb 40-60, Houttuynia cordata 200- 300, honeysuckle 200-300, Banlangen 200-300, patchouli 60-100, Mianma Guanzhong 200-300, Rhodiola 60-100, menthol 5-9, bitter almond 60-100, licorice 60-100 , gypsum 200-300;

The content determination method is:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, grind finely, accurately weigh it, place it in a conical flask, add 25mL of ethanol or methanol, extract by ultrasonic or reflux for 30 minutes, filter, obtain filtrate, and dry it. , the residue is dissolved in ethanol or methanol and made up to 25mL, that is;

Preparation of reference solution: taking fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate reference substance Appropriate amount, accurately weighed, add 50% methanol to make 34.4μg of new chlorogenic acid per 1mL, 76.7μg chlorogenic acid, 34.2μg cryptochlorogenic acid, 56.2μg of allosperglycin A, 14.9μg4,5-di- a mixed solution of O-caffeoylquinic acid, 11.2 μg of quercetin and 42.8 μg of ammonium glycyrrhizinate, wherein, the weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207;

Chromatographic conditions: methanol-acetonitrile-0.1% phosphoric acid solution as mobile phase, gradient elution procedure is:

The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; the column is a C 18 column;

Determination method: respectively, accurately draw 2 μL of the reference solution and the test solution, inject into the liquid chromatograph, measure and record the chromatogram, that is, obtain.
The content determination method according to claim 5, wherein the traditional Chinese medicine composition is made of the following raw materials by weight:

Forsythia 200, Honeysuckle 300, Banlangen 200, Rhubarb 40, Patchouli 60, Mianma Guanzhong 300, Rhodiola 100, Menthol 9, Ephedra 60, Bitter Almond 100, Houttuynia 200, Licorice 100, Gypsum 200 .
The content determination method according to claim 5, wherein the traditional Chinese medicine composition is made of the following raw materials by weight:

Forsythia 300, Honeysuckle 200, Banlangen 300, Rhubarb 60, Patchouli 100, Mianma Guanzhong 200, Rhodiola 60, Menthol 5, Ephedra 100, Bitter Almond 60, Houttuynia 300, Licorice 60, Gypsum 300 .
The content determination method according to claim 5, wherein the traditional Chinese medicine composition is made of the following raw materials by weight:

Forsythia 278, Honeysuckle 294, Banlangen 285, Rhubarb 55, Patchouli 95, Mianma Guanzhong 290, Rhodiola 87, Menthol 8.5, Ephedra 88, Bitter Almond 80, Houttuynia 284, Licorice 95, Gypsum 277 .
The content determination method according to claim 5, wherein the traditional Chinese medicine composition is made of the following raw materials by weight:

Forsythia 255, Honeysuckle 255, Banlangen 255, Rhubarb 51, Patchouli 85, Mianma Guanzhong 255, Rhodiola 85, Menthol 7.5, Ephedra 85, Bitter Almond 85, Houttuynia 255, Licorice 85, Gypsum 255 .
The content determination method according to any one of claims 1 to 9, wherein the traditional Chinese medicine composition is produced by the following steps:

(1) Weigh Chinese medicines according to the weight ratio of raw materials, net selection, and break as appropriate;

(2) Patchouli scented, add 10 times the amount of water to extract volatile oil, oil extraction time 8 hours, collect volatile oil, spare; after the extract is filtered, the residue is discarded, the filtrate is reserved;

(3) Forsythia, Ephedra, Houttuynia cordata, Rhubarb, extracted with 12 times 70% ethanol for 3 times, each time for 2.5 hours, the extract is combined with filtration, ethanol is recovered, and the filtrate is reserved;

(4) Honeysuckle, gypsum, Banlangen, Mianma Guanzhong, Licorice, Rhodiola, add 12 times the amount of water to boil, add bitter almonds, decoct 2 times, each time for 1 hour, extract and filter, the result The filtrate is combined with the filtrate of step (2) patchouli oil extraction, and concentrated to obtain a clear paste having a relative density of 1.10-15.15 at 60 ° C, added with ethanol, adjusted to an alcohol concentration of 70%, refrigerated, filtered, and recovered. Ethanol to no alcohol, get a clear paste;

(5) Combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), and concentrating to obtain a clear paste having a relative density of 1.15 - 1.20 at 60 ° C, and drying to obtain a dry paste powder, which is used;

(6) adding the dry paste powder obtained in the step (5) to a suitable pharmaceutically acceptable auxiliary material for granulation;

(7) The menthol and the volatile oil obtained in the step (2) are dissolved in ethanol, and the granules obtained in the step (6) are sprayed, sealed, mixed, tableted or capsuled, or bagged.
The content determination method according to any one of claims 1 to 9, wherein the content determination method is:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 50mL ethanol, 25mL, ultrasonic extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter, The filtrate was obtained, and the filter paper was added together with the residue into a stoppered conical flask, and 25 mL of 50% ethanol was added thereto, sonicated for 30 minutes, filtered, and the dregs were washed 4 times with 50% ethanol, 5 mL each time, and the filtrate and washing were combined twice. The liquid is evaporated to dryness, and the residue is dissolved in 50% methanol and made up to 25 mL, which is obtained;

Preparation of reference solution: taking fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate reference substance Appropriate amount, accurately weighed, add 50% methanol to make 34.4μg of new chlorogenic acid per 1mL, 76.7μg chlorogenic acid, 34.2μg cryptochlorogenic acid, 56.2μg of allosperglycin A, 14.9μg4,5-di- a mixed solution of O-caffeoylquinic acid, 11.2 μg of quercetin and 42.8 μg of ammonium glycyrrhizinate, wherein, the weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207;

Chromatographic conditions: methanol-acetonitrile-0.1% phosphoric acid solution as mobile phase, gradient elution procedure is:

The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; column: ACQUITY
T3 (1.8 μm, 2.1×100 mm); the column temperature is 35 ° C;

Determination method: respectively, accurately draw 2 μL of the reference solution and the test solution, inject into the liquid chromatograph, measure and record the chromatogram, that is, obtain.
The content determination method according to any one of claims 1 to 9, wherein the content determination method is:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, grind finely, accurately weigh it, place it in a conical flask, add 50% methanol 25mL, ultrasonic extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter The filtrate was obtained, and the filter paper was added together with the residue into a stoppered conical flask, and then 50 mL of 50% methanol was added thereto, sonicated for 30 minutes, filtered, and the dregs were washed 4 times with 50% methanol, 5 mL each time, and the filtrate was combined twice. The washing liquid is evaporated to dryness, and the residue is dissolved in 50% methanol and made up to 25 mL, which is obtained;

Preparation of reference solution: taking fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate reference substance Appropriate amount, accurately weighed, add 50% methanol to make 34.4μg of new chlorogenic acid per 1mL, 76.7μg chlorogenic acid, 34.2μg cryptochlorogenic acid, 56.2μg of allosperglycin A, 14.9μg4,5-di- a mixed solution of O-caffeoylquinic acid, 11.2 μg of quercetin and 42.8 μg of ammonium glycyrrhizinate, wherein, the weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207;

Chromatographic conditions: methanol-acetonitrile-0.1% phosphoric acid solution as mobile phase, gradient elution procedure is:

The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; column is ACQUITY

Determination method: respectively, accurately draw 2 μL of the reference solution and the test solution, inject into the liquid chromatograph, measure and record the chromatogram, that is, obtain.
The content determination method according to any one of claims 1 to 9, wherein the content determination method is:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 25mL of methanol, reflux extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter, and obtain filtrate Add the filter paper together with the residue into a stoppered conical flask, add 25mL of methanol, sonicate for 30 minutes, filter, wash the dregs with methanol 4 times, each time 5mL, combine the filtrate and washing solution twice, evaporate and dry, residue Dissolve with 50% methanol and dilute to 25mL, that is;

Preparation of reference solution: taking fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate The appropriate amount of the reference substance, accurately weighed, added 50% methanol to make 34.4 μg of fresh chlorogenic acid per 1 mL, 76.7 μg of chlorogenic acid, 34.2 μg of cryptochlorogenic acid, 56.2 μg of allosperglycin A, 14.9 μg 4,5- a mixed solution of di-O-caffeoylquinic acid, 11.2 μg of quercetin and 42.8 μg of ammonium glycyrrhizinate, wherein, the weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207;

Chromatographic conditions: methanol-acetonitrile-0.1% phosphoric acid solution as mobile phase, gradient elution procedure is:

The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; column is ACQUITY
C 18 ;

Determination method: respectively, accurately draw 2 μL of the reference solution and the test solution, inject into the liquid chromatograph, measure and record the chromatogram, that is, obtain.
The content determination method according to any one of claims 1 to 9, wherein the content determination method is:

Preparation of test solution: Take 0.5g of the traditional Chinese medicine composition, finely weigh it, place it in a conical flask, add 25mL of ethanol, ultrasonic extraction, power 500W, frequency 40kHz, extract for 30 minutes, filter, and obtain filtrate Add the filter paper together with the residue into the stoppered conical flask, add 25mL of ethanol, sonicate for 30 minutes, filter, wash the dregs with ethanol 4 times, each time 5mL, combine the filtrate and washing solution twice, evaporate and dry, residue Dissolve with 50% methanol and dilute to 25mL, that is;

Preparation of reference solution: taking fresh chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A, 4,5-di-O-caffeoyl quinic acid, quercetin, ammonium glycyrrhizinate reference substance Appropriate amount, accurately weighed, add 50% methanol to make 34.4μg of new chlorogenic acid per 1mL, 76.7μg chlorogenic acid, 34.2μg cryptochlorogenic acid, 56.2μg of allosperglycin A, 14.9μg4,5-di- a mixed solution of O-caffeoylquinic acid, 11.2 μg of quercetin and 42.8 μg of ammonium glycyrrhizinate, wherein, the weight of glycyrrhizic acid = weight of ammonium glycyrrhizinate / 1.0207;

Chromatographic conditions: methanol-acetonitrile-0.1% phosphoric acid solution as mobile phase, gradient elution procedure is:

The detection wavelengths were 327 nm and 250 nm respectively; quercetin and glycyrrhizic acid were detected at 250 nm, and new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isophoric acid A and 4,5-di-O-coffee were detected at 327 nm. Acyl quinic acid; column is ACQUITY
Shield RP 18;

Determination method: respectively, accurately draw 2 μL of the reference solution and the test solution, inject into the liquid chromatograph, measure and record the chromatogram, that is, obtain.