CN104345108A

CN104345108A - Qualitative quantitative determination method for liver-heat-clearing tablet

Info

Publication number: CN104345108A
Application number: CN201310319584.4A
Authority: CN
Inventors: 郗瑞云; 谷陟欣; 张妮瑜; 欧金秀; 辛秀; 刘淑妦; 袁莉; 朱丽
Original assignee: Jiuzhitang Co Ltd
Current assignee: Jiuzhitang Co Ltd
Priority date: 2013-07-29
Filing date: 2013-07-29
Publication date: 2015-02-11
Anticipated expiration: 2033-07-29
Also published as: CN104345108B

Abstract

The invention discloses a qualitative quantitative determination method for a liver-heat-clearing tablet, and a conventional qualitative identification method for herba artemisiae scopariae in the liver-heat-clearing tablet is improved. The following steps are increased: (1) employing a microscopic identification process to identify radix isatidis in the liver-heat-clearing tablet; (2) employing thin-layer chromatography to identify radix isatidis in the liver-heat-clearing tablet by taking indirubin as a reference; and (3) employing high performance liquid chromatography to determine the content of chlorogenic acid in herba artemisiae scopariae and the content of glycyrrhizic acid in glycyrrhiza. Compared with the prior art, in the method, a qualitative discriminating method is added and also the content determination method for the effective compositions is added. The method is high in precision, good in reproducibility, high in recovery rate and accurate in determination result, and helps to improve the quality controllability and stability of the liver-heat-clearing tablet, and guarantee people drug use safety and validity. The qualitative quantitative determination method for the liver-heat-clearing tablet is capable of controlling the quality of the liver-heat-clearing tablet, also is capable of controlling the quality of other dosage forms of a same prescription, such as a capsule, a granule and the like.

Description

A kind of method for qualitative and quantitative detection of Qinggan Tablet

Technical field

The invention belongs to technical field of traditional Chinese medicines, be specifically related to a kind of method of quality control of Qinggan Tablet.

Background technology

Qinggan Tablet is made up of Radix Isatidis, oriental wormwood, Radix Glycyrrhizae, has clearing heat and detoxicating, and effect of dehumidifying cholagogic, can be used for cold, fever, abscess of throat, mumps, the illnesss such as jaundice with damp-heat pathogen.The quality standard of this product is recorded in " Drug Standard of Ministry of Public Health of the Peoples Republic of China " Traditional Chinese medicine historical preparation the 3rd, standard number WS ₃-B-0626-91, only has the qualitative identification method of oriental wormwood and Radix Glycyrrhizae in this standard, without any quantitative detecting method, be unfavorable for the product quality controlling Qinggan Tablet.Qinggan Tablet has been recorded in country's standard for traditional Chinese medicines compilation internal medicine liver and gall fascicle, oriental wormwood, the qualitative identification method of Radix Glycyrrhizae and the content assaying method of glycyrrhizic acid is had in this standard, but Qualitive test was not both carried out to monarch drug in a prescription Radix Isatidis and there is no quantitative examination yet, and only had the assay of glycyrrhizic acid 1 effective ingredient.Time precious traditional Chinese medical science traditional Chinese medicines the 18th volume the 1st phase " quality standard research of Qinggan Tablet " in 2007 describe the method for quality control of Qinggan Tablet, Qualitive test research has been carried out to Radix Isatidis, oriental wormwood, Radix Glycyrrhizae, assay has been carried out to glycyrrhizic acid, but microscopical characters is not carried out to the Radix Isatidis that former powder is used as medicine, Radix Isatidis and oriental wormwood separate to differentiate, and only have the assay of 1 effective ingredient.Existing quality standard effectively can not control the quality of Qinggan Tablet, thus will affect the production of this product and quality assurance and clinical efficacy thereof.

Summary of the invention

The object of the invention is to: the method for qualitative and quantitative detection that a kind of Qinggan Tablet is provided.The present invention is directed to the deficiency of existing control criterion, the method for quality control of Qinggan Tablet is studied, improve the Qualitative Identification method of oriental wormwood in original Qinggan Tablet, add: (1) adopts Microscopic Identification method to differentiate Radix Isatidis in Qinggan Tablet; (2) adopt thin-layered chromatography, with oriental wormwood medicinal material for control medicinal material, take indigo red as contrast, differentiate in Qinggan Tablet containing oriental wormwood and Radix Isatidis; (3) content of high effective liquid chromatography for measuring oriental wormwood Content of Chlorogenic Acid and Radix Glycyrrhizae acid is adopted.Method for qualitative and quantitative detection provided by the present invention, improves the quality control standard of Qinggan Tablet, thus ensure that quality and the clinical efficacy of this medicine.

The present invention is achieved in that

A method for qualitative and quantitative detection for Qinggan Tablet, comprises the Qualitive test of the thin-layered chromatography of Radix Glycyrrhizae, also comprises and 1. adopts Microscopic Identification method to differentiate Radix Isatidis in Qinggan Tablet; 2. adopt thin-layered chromatography, with oriental wormwood medicinal material for control medicinal material, take indigo red as contrast, differentiate in Qinggan Tablet containing oriental wormwood and Radix Isatidis; 3. the content of high effective liquid chromatography for measuring oriental wormwood Content of Chlorogenic Acid and Radix Glycyrrhizae acid is adopted; Described method for qualitative and quantitative detection comprises following steps:

(1) get this product, removing dressing, porphyrize, adds water saturated normal butyl alcohol, constantly jolting, and filter, filtrate puts evaporate to dryness in water-bath, and residue adds methyl alcohol and dissolves, as need testing solution; Another extracting Radix Glycyrrhizae control medicinal material 0.3 ~ 1g, is made in the same way of control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 ~ 15 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-ethanol-ammonia solution=4 ~ 6:1.5 ~ 2.5:0.5 ~ 1.5 for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development in 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material, the spot of aobvious same color;

(2) get this product powder, thoroughly change rear load, put basis of microscopic observation: reticulate vessel is more common with chloral hydrate, mesh is shorter, conduit diameter 7 ~ 51 μm; Many bunchys of xylogen or to be singlely dispersed in, diameter 14 ~ 20 μm, micro-lignify, pit and hole ditch obvious;

(3) get this product, removing dressing, porphyrize, adds ethanol, ultrasonic process, and filter, filtrate evaporate to dryness, residue adds ethanol makes dissolving, as need testing solution.Separately get oriental wormwood control medicinal material 1 ~ 3g, be made in the same way of oriental wormwood control medicinal material solution.Get indigo red reference substance again, add ethanol and make solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 ~ 15 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=5 ~ 9:0.2 ~ 0.7:1.5 ~ 3.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color;

(4) glycyrrhizic acid content measures: measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D);

Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile-0.01 ~ 0.03mol/L phosphoric acid solution (30 ~ 50:70 ~ 50) is mobile phase; Flow velocity is 0.5 ~ 1.0mL/min; Determined wavelength is 250nm; Column temperature 25 ~ 35 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

The preparation of reference substance solution: extracting Radix Glycyrrhizae acid mono-ammonium reference substance is appropriate, adds 70% ethanol and makes the solution of every 1ml containing ammonium glycyrrhetate 40 μ g, obtain (amounting to glycyrrhizic acid is 39.2 μ g);

The preparation of need testing solution: get this product, removing dressing, porphyrize, put in tool plug conical flask, precision adds 50 ~ 80% ethanol, weighed weight, at power 250W, under frequency 50kHz condition, ultrasonic process, lets cool, and supplies the weight of less loss with 50 ~ 80% ethanol, shake up, filter with filter membrane, get subsequent filtrate, to obtain final product;

Determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product;

The every sheet of this product contains Radix Glycyrrhizae with glycyrrhizic acid (C ₄₂h ₆₂o ₁₆) meter, must not 0.4mg be less than;

(5) determination of chlorogenic acid: measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D):

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile-0.01 ~ 0.03mol/L phosphoric acid solution (5 ~ 15:95 ~ 85) is mobile phase; Flow velocity is 0.5 ~ 1.0mL/min; Determined wavelength is 327nm; Column temperature 25 ~ 35 DEG C; Number of theoretical plate calculates should be not less than 5000 by chlorogenic acid peak;

The preparation of reference substance solution gets chlorogenic acid reference substance in right amount, puts in brown measuring bottle, adds 70% ethanol and makes the solution of every 1ml containing chlorogenic acid 0.01mg; Obtain;

The every sheet of this product contains oriental wormwood with chlorogenic acid (C ₁₆h ₁₈o ₉) meter, must not 0.12mg be less than.

To better implement the present invention, Qinggan Tablet of the present invention method for qualitative and quantitative detection, can be optimized for further:

(1) get this product 5 ~ 15, removing dressing, porphyrize, adds water saturated normal butyl alcohol 5 ~ 15ml, places 0.5 ~ 1.5 hour, jolting constantly, and filter, filtrate puts evaporate to dryness in water-bath, and residue adds methyl alcohol 1 ~ 3ml and dissolves, as need testing solution; Another extracting Radix Glycyrrhizae control medicinal material 0.3 ~ 1g, is made in the same way of control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 ~ 15 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-ethanol-ammonia solution=4 ~ 6:1.5 ~ 2.5:0.5 ~ 1.5 for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development in 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material, the spot of aobvious same color;

(3) get this product 5 ~ 15, removing dressing, porphyrize, adds ethanol 15 ~ 50ml, ultrasonic process 30 ~ 60 minutes, and filter, filtrate evaporate to dryness, residue adds ethanol 1 ~ 3ml makes dissolving, as need testing solution.Separately get oriental wormwood control medicinal material 1 ~ 3g, be made in the same way of oriental wormwood control medicinal material solution.Get indigo red reference substance again, add ethanol and make the solution containing 0.05 ~ 0.2mg in every 1ml.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 ~ 15 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=5 ~ 9:0.2 ~ 0.7:1.5 ~ 3.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color;

Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile: 0.01 ~ 0.03mol/L phosphoric acid solution (30 ~ 50:70 ~ 50) is mobile phase; Flow velocity is 0.5 ~ 1.0mL/min; Determined wavelength is 250nm; Column temperature 25 ~ 35 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

The preparation of need testing solution: get this product 5 ~ 20, removing dressing, porphyrize, get about 0.2 ~ 1.0g, put in tool plug conical flask, precision adds 50 ~ 80% ethanol 20 ~ 50ml, weighed weight, ultrasonic process under power 250W, frequency 50kHz condition, let cool, supply the weight of less loss with 50 ~ 80% ethanol, shake up, filter with filter membrane, get subsequent filtrate, to obtain final product;

To better implement the present invention, Qinggan Tablet of the present invention method for qualitative and quantitative detection, can be optimized for further again:

(1) get this product 10, removing dressing, porphyrize, adds water saturated normal butyl alcohol 10ml, places 1 hour, jolting constantly, and filter, filtrate puts evaporate to dryness in water-bath, and residue adds methyl alcohol 1ml and dissolves, as need testing solution; Another extracting Radix Glycyrrhizae control medicinal material 0.3g, is made in the same way of control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-ethanol-ammonia solution=5:2:1 for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development in 105 DEG C clear; In test sample chromatogram, on the position corresponding to control medicinal material, the spot of aobvious same color;

(3) get this product 10, removing dressing, porphyrize, adds ethanol 20ml, ultrasonic process 30 minutes, and filter, filtrate evaporate to dryness, residue adds ethanol 2ml makes dissolving, as need testing solution.Separately get oriental wormwood control medicinal material 1.5g, be made in the same way of oriental wormwood control medicinal material solution.Get indigo red reference substance again, add ethanol and make the solution containing 0.1mg in every 1ml.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=7:0.5:2.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color;

Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile: 0.017mol/L phosphoric acid solution (35:65) is mobile phase; Flow velocity is 0.7mL/min; Determined wavelength is 250nm; Column temperature 30 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

The preparation of need testing solution: get this product 20, removing dressing, porphyrize, get about 0.5g, put in tool plug conical flask, precision adds 70% ethanol 25ml, weighed weight, ultrasonic process under power 250W, frequency 50kHz condition, let cool, supply the weight of less loss with 70% ethanol, shake up, filter with 0.45 μm of filter membrane, get subsequent filtrate, to obtain final product;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile: 0.017mol/L phosphoric acid solution (10:90) is mobile phase; Flow velocity is 0.8mL/min; Determined wavelength is 327nm; Column temperature 30 DEG C; Number of theoretical plate calculates should be not less than 5000 by chlorogenic acid peak;

For better showing essence of the present invention, by methodological study of the present invention, details are as follows:

1, get this product powder, thoroughly change rear load, put basis of microscopic observation: reticulate vessel is more common with chloral hydrate, mesh is shorter, conduit diameter 7 ~ 51 μm; Many bunchys of xylogen or to be singlely dispersed in, diameter 14 ~ 20 μm, micro-lignify, pit and hole ditch obvious.See Fig. 1.

2, get this product 10, removing dressing, porphyrize, adds ethanol 20ml, ultrasonic process 30 minutes, and filter, filtrate evaporate to dryness, residue adds ethanol 2ml makes dissolving, as need testing solution.Take other medicinal materials of scarce oriental wormwood by prescription, make the negative control sample of scarce oriental wormwood, be made in the same way of the negative control solution of scarce oriental wormwood.Take other medicinal materials of scarce Radix Isatidis by prescription, make the negative control sample of scarce Radix Isatidis, be made in the same way of the negative control solution of scarce Radix Isatidis.Separately get oriental wormwood control medicinal material 1.5g, be made in the same way of oriental wormwood control medicinal material solution.Get indigo red reference substance again, add ethanol and make the solution containing 0.1mg in every 1ml.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ l of above-mentioned five kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=7:0.5:2.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color.See Fig. 2,3.

3, get this product 10, removing dressing, porphyrize, adds water saturated normal butyl alcohol 10ml, places 1 hour, jolting constantly, and filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Take other medicinal materials of scarce Radix Glycyrrhizae by prescription, make the negative control sample of scarce Radix Glycyrrhizae, be made in the same way of the negative control solution of scarce Radix Glycyrrhizae.Another extracting Radix Glycyrrhizae control medicinal material 0.3g, is made in the same way of control medicinal material solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-ethanol-ammonia solution (5: 2: 1) for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.See Fig. 4.

4, glycyrrhizic acid content measures: measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D);

(1) instrument and reagent: Waters1525 high performance liquid chromatograph, Wasters 2487 detecting device, AY220 electronic analytical balance, CP225D electronic analytical balance, C ₁₈post Inertsil ODS-2 (250mm × 4.6mm, 5 μm), column temperature 30 DEG C, mobile phase acetonitrile-0.017mol/L phosphoric acid solution (35: 65), flow velocity is 0.7ml/min, Empower2 chromatographic work station; Acetonitrile is chromatographically pure, and it is pure that other reagent are analysis.

Ammonium glycyrrhetate (Nat'l Pharmaceutical & Biological Products Control Institute provides, for assay, and lot number: 110731-200614).

(2) preparation of reference substance solution:

Extracting Radix Glycyrrhizae acid mono-ammonium reference substance is appropriate, adds 70% ethanol and makes the solution of every 1ml containing ammonium glycyrrhetate 40 μ g, obtain (amounting to glycyrrhizic acid is 39.2 μ g);

(3) selection of mobile phase

When adopting the content of high effective liquid chromatography for measuring glycyrrhizic acid, conventional mobile phase has: 1. acetonitrile-0.017mol/L phosphoric acid solution (35:65), 2. methanol-water-acetic acid (60:30:5).Respectively with the test of two kinds of mobile phases, the number of theoretical plate of mobile phase 1. gained glycyrrhizic acid chromatographic peak is high, peak symmetry better, therefore selects acetonitrile-0.017mol/L phosphoric acid solution (35:65) to be mobile phase.In table 1.

Table 1 mobile phase selection result comparison sheet

(4) selection of wavelength is measured

Extracting Radix Glycyrrhizae acid reference substance solution, in the interscan of 200 ~ 400nm wavelength coverage, result glycyrrhizic acid has absorption maximum at 250.0 nm wavelength places, therefore selects determined wavelength to be 250 nm, sees Fig. 5.

(5) selection of Extraction solvent

Get above-mentioned powder and be about 0.5g, totally three parts, accurately weighed, put in tool plug conical flask, precision adds 50% ethanol, 70% ethanol, methyl alcohol 25ml successively, close plug, weighed weight, ultrasonic process (300W, 33kHz) 30 minutes, let cool, add the weight that coordinative solvent supplies less loss respectively, shake up, filter, get subsequent filtrate, to obtain final product.Accurate absorption reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measure and calculate, result shows, with 3 kinds of solvent extraction test samples, it is higher that 70% ethanol records content results, therefore, selects 70% ethanol to be Extraction solvent.In table 2.

Table 2 Extraction solvent selection result comparison sheet

(6) investigation of extracting method, extraction time

Get Qinggan Tablet sample powder and be about 0.5g, totally 8 parts, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 25ml, close plug, weigh, respectively ultrasonic process (300W, 33kHz) 15,30,45,60 minutes, let cool, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.

Get Qinggan Tablet sample powder about 0.5 g, totally 2 parts, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 25ml, close plug, weighed weight, and refluxing extraction 30 minutes, lets cool, and supplies the weight of less loss, shake up with 70% ethanol, filters, gets subsequent filtrate, to obtain final product.

Determination method precision draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, and measure, and calculate, result shows, refluxing extraction is close with ultrasonic extraction yield, because ultrasonic processing method is easier, therefore selects ultrasonic extraction.Ultrasonic process 30 minutes yield are the highest, therefore extraction time is chosen as 30 minutes.In table 3.

Table 3 extracting method, selection of time results contrast table

Comprehensive above-mentioned investigation result, the preparation method of need testing solution is defined as: get this product 10, removing dressing, accurately weighed, porphyrize, gets about 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 25ml, close plug, weigh, ultrasonic process (300W, 33kHz) 30 minutes, let cool, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.

(7) specificity test takes all the other each flavour of a drug of scarce Radix Glycyrrhizae in right amount by prescription, makes the negative control sample of scarce Radix Glycyrrhizae, with the standby negative control solution lacking Radix Glycyrrhizae of legal system, measures in accordance with the law.Result: in the chromatogram of negative control, at the identical retention time place of glycyrrhizic acid reference substance chromatographic peak without chromatographic peak, illustrates that negative control is noiseless.See Fig. 6,7,8.

(8) replica test gets same lot number (J0002) sample, prepares 6 parts of need testing solutions in accordance with the law, and measure, result shows: this method repeatability better.In table 4.

Table 4 replica test result

(9) precision test precision draws same need testing solution (lot number: J0002), sample introduction 6 times, each 10 μ l, and measure, the RSD of its glycyrrhizic acid integrating peak areas value is 0.13%, and result shows that sample introduction precision is good.In table 5.

Table 5 sample introduction Precision test result table

(10) same need testing solution is got in stability test, and respectively at 0,2,4,8,16,24 hour after preparation, accurate draw 10 μ l, injection liquid chromatography, measure, result shows: need testing solution is stable in latter 24 hours of preparation.In table 6.

Table 6 stability test result table

(11) linear relationship is investigated

Get the glycyrrhizic acid solution of 0.2102 mg/ml, successively in accurate absorption 0.5ml, 1ml, 2ml, 4ml, 6ml to a five 10ml volumetric flask, add 70% ethanol to scale, shake up, obtain control series product solution (0.01051,0.02102,0.04204,0.08408,0.1261,0.2102 mg/ml).The accurate reference substance solution drawing above-mentioned 6 variable concentrations of 10 μ l respectively, injection liquid chromatography, measure, with integrating peak areas value for ordinate, sample size is (μ g) is horizontal ordinate, drawing standard curve.Its regression equation is: Y=1106389.2869X-7582.4435, r=0.9998.Result shows: glycyrrhizic acid reference substance is good in 0.1051 ~ 2.102 μ g scope internal linear relation.In table 7, Fig. 9.

Table 7 linear relationship investigates result table

(12) detectability precision draws glycyrrhizic acid solution (0.8408 μ g/ml) 1 μ l, injection liquid chromatography, and measure, signal to noise ratio (S/N ratio) is about 3, therefore detection is limited to 0.8408 ng(Figure 14).

(13) quantitative limit precision draws glycyrrhizic acid solution (0.8408 μ g/ml) 3 μ l, injection liquid chromatography, and measure, signal to noise ratio (S/N ratio) is about 10, therefore is quantitatively limited to 2.522 ng.METHOD FOR CONTINUOUS DETERMINATION 6 times, its integrating peak areas value RSD is 1.09%, the results are shown in Table 8.

Table 8 quantitative limit test findings table

(14) same lot number (J0002) sample (containing the glycyrrhizic acid 1.8246 mg/ sheet) 0.25g that 6 parts have measured content is got in average recovery test, accurately weighed respectively, after precision adds 0.2102 mg/ml glycyrrhizic acid solution 2 ml, precision adds 70% ethanol 25ml again, and close plug, weighs, ultrasonic process (300W, 33kHz) 30 minutes, lets cool, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, obtain need testing solution, measure also result of calculation and show, this law recovery is good.In table 9.

Table 9 recovery test result

(15) durability is investigated

Adopt the method drafted, chromatographic column, the high performance liquid chromatograph of different 2 different brands of employing measure same batch sample, and measurement result is in table 10, table 11.

Table 10 instrument and chromatographic column model

Table 11 serviceability test result

Result shows, this method good tolerance.

(16) sample size measures

Assay is carried out to 10 batches of Qinggan Tablets, the results are shown in Table 12.

Glycyrrhizic acid content measurement result in table 12 Qinggan Tablet

5, determination of chlorogenic acid: measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D):

(1) instrument and reagent

Wasters 1525 high performance liquid chromatograph, Wasters 2487 detecting device, AY220 electronic analytical balance, CP225D electronic analytical balance, C ₁₈post Inertsil ODS-2 (250mm × 4.6mm, 5 μm), column temperature 30 DEG C, mobile phase acetonitrile-0.017mol/L phosphoric acid solution (10: 90), flow velocity is 0.8ml/min, Empower2 chromatographic work station; Acetonitrile is chromatographically pure, and it is pure that other reagent are analysis.

Chlorogenic acid (Nat'l Pharmaceutical & Biological Products Control Institute provides, for assay, and lot number: 110753-200413).

(2) preparation of reference substance solution

Get chlorogenic acid reference substance appropriate, put in brown measuring bottle, add 70% ethanol and make the solution of every 1ml containing chlorogenic acid 0.01mg; Obtain;

(3) selection of mobile phase

When adopting the content of high effective liquid chromatography for measuring chlorogenic acid, conventional mobile phase has: 1. acetonitrile-0.017mol/L phosphoric acid solution (10:90), 2. methanol-water-glacial acetic acid (20:80:1).Respectively with the test of two kinds of mobile phases, the number of theoretical plate of mobile phase 1. gained chlorogenic acid chromatographic peak is high, peak symmetry better, therefore selects acetonitrile-0.017mol/L phosphoric acid solution (10:90) to be mobile phase.In table 13.

Table 13 mobile phase selection result comparison sheet

(4) selection of wavelength is measured

Get reference substance solution in the interscan of 200 ~ 800nm wavelength coverage, result chlorogenic acid has absorption maximum at 326nm wavelength place, and the determined wavelength in conjunction with pharmacopeia chlorogenic acid mostly is 327nm, therefore selects determined wavelength to be 327nm.See Figure 10.

(5) selection of Extraction solvent

Get above-mentioned powder and be about 0.5g, totally 6 parts, accurately weighed, put in tool plug conical flask, be divided into 3 groups, often organize 2 parts, precision adds methyl alcohol, 50% methyl alcohol, 70% ethanol 25ml successively, close plug, weighed weight, ultrasonic process (300W, 33kHz) 30 minutes, lets cool, add the weight that coordinative solvent supplies less loss respectively, shake up, filter, get subsequent filtrate, to obtain final product.Accurate absorption reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measure and calculate, result shows, with 3 kinds of solvent extraction test samples, 50% methyl alcohol is suitable with the extraction efficiency of 70% ethanol to chlorogenic acid, consider that alcohol toxicity is little, and shared same test sample can be measured with glycyrrhizic acid content, simplify experimental procedure, therefore select 70% ethanol to be Extraction solvent.In table 14.

Table 14 Extraction solvent selection result comparison sheet

(6) investigation of extraction time

Get Qinggan Tablet sample powder and be about 0.5g, totally 8 parts, be divided into 4 groups, often organize 2 parts, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 25ml, and close plug, weighs, ultrasonic process (300W, 33kHz) 15,30,45,60 minutes, lets cool respectively, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.

Determination method precision draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, and measure, and calculate, result shows, the chlorogenic acid yield that ultrasonic process obtains for 30 minutes is the highest, therefore determines that the optimum extraction time is 30 minutes.In table 15.

Table 15 extraction time selection result comparison sheet

(7) investigation of extracting mode

Get Qinggan Tablet sample powder and be about 0.5g, totally 4 parts, be divided into 2 groups, often organize 2 parts, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 25ml, and close plug, weighs, respectively ultrasonic process (300W, 33kHz) 30 minutes and heating and refluxing extraction 30 minutes, let cool, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.

Determination method precision draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measure, and calculate, result shows, ultrasonic process 30 minutes, with to add the chlorogenic acid yield that hot reflux obtains for 30 minutes suitable, be simplify test sample treatment step, therefore determine that optimum extraction mode is ultrasonic 30 minutes.In table 16.

Table 16 extracting mode selection result comparison sheet

(8) specificity test takes all the other each flavour of a drug of scarce oriental wormwood in right amount by prescription, makes the negative control sample of scarce oriental wormwood, with the standby negative control solution lacking oriental wormwood of legal system, measures in accordance with the law.Result: in the chromatogram of negative control, at the identical retention time place of chlorogenic acid contrast chromatographic peak without chromatographic peak, illustrates that negative control is noiseless.See Figure 11,12,13.

(9) replica test gets same lot number (J0002) sample, prepares 6 parts of need testing solutions in accordance with the law, and measure, result shows: this method repeatability better.In table 17.

Table 17 replica test result

(10) precision test precision draws same need testing solution (lot number: J0002), sample introduction 6 times, each 10 μ l, and measure, the RSD of its chlorogenic acid integrating peak areas value is 0.43%, and result shows that sample introduction precision is good.In table 18.

Table 18 sample introduction Precision test result table

(11) same need testing solution is got in stability test, and respectively at 0,2,4,12,16,24 hour after preparation, accurate absorption 10 μ l, injection liquid chromatography, measured.Result shows: need testing solution is stable in latter 24 hours of preparation.In table 19.

Table 19 stability test result table

(12) linear relationship is investigated

Get the solution of chlorogenic acid of 0.1230 mg/ml, successively in accurate absorption 1ml, 2ml, 3ml to a three 25ml volumetric flask, precision is drawn in 3ml, 5ml to two 10ml volumetric flask successively again, add 70% ethanol respectively to scale, shake up, obtain control series product solution (0.00492,0.00984,0.01476,0.03690,0.06150,0.1230 mg/ml).The accurate reference substance solution drawing above-mentioned 6 variable concentrations of 10 μ l respectively, injection liquid chromatography, measure, with integrating peak areas value for ordinate, sample size (μ g) is horizontal ordinate, drawing standard curve.Its regression equation is: Y=3143503.1801X+9411.8034, r=0.9999.Result shows: chlorogenic acid reference substance is good in 0.0492 ~ 1.23 μ g scope internal linear relation.In table 20, Figure 14.

Table 20 linear relationship investigates result table

(13) detectability gets solution of chlorogenic acid (0.004920 mg/ml), and precision measures 1ml to 100ml measuring bottle, adds 50% methyl alcohol to scale, shakes up, and obtains the solution that chlorogenic acid concentration is 0.04920 μ g/ml.The accurate solution of chlorogenic acid 4 μ l drawing 0.04920 μ g/ml, injection liquid chromatography, measure, signal to noise ratio (S/N ratio) is about 3, therefore detection is limited to 0.1968 ng.

(14) quantitative limit precision draws solution of chlorogenic acid (0.04920 μ g/ml) 12 μ l, injection liquid chromatography, and measure, signal to noise ratio (S/N ratio) is about 10, therefore is quantitatively limited to 0.5904 ng.METHOD FOR CONTINUOUS DETERMINATION 6 times, its integrating peak areas value RSD is 2.30%, the results are shown in Table 21.

Table 21 quantitative limit test findings table

(15) same lot number (J0002) sample (containing the chlorogenic acid 0.4722 mg/g) 0.25g that 6 parts have measured content is got in average recovery test, accurately weighed respectively, after every part of precision adds 0.1230 mg/ml solution of chlorogenic acid 1ml, precision adds 70% ethanol 25ml again, close plug, weighs, ultrasonic process (300W, 33kHz) 30 minutes, let cool, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, obtain need testing solution, measure and calculate, result shows, this law recovery is good.In table 22.

Table 22 recovery test result

(16) durability is investigated

Adopt the method drafted, chromatographic column, the high performance liquid chromatograph of different 2 different brands of employing measure same batch sample, and result shows, this method good tolerance.In table 23,24.

Table 23 instrument and chromatographic column model

Table 24 serviceability test result

(17) sample size measures

Assay is carried out to 10 batch sample, the results are shown in Table 25.

Table 25 Qinggan Tablet Content of Chlorogenic Acid measurement result

Beneficial effect of the present invention:

(1) respectively microscopical characters and indentification by TLC have been carried out to monarch drug in a prescription Radix Isatidis, and Radix Isatidis and oriental wormwood differentiate that test sample preparation method is consistent with development system;

(2) assay has been carried out to the glycyrrhizic acid in Radix Glycyrrhizae in Qinggan Tablet and oriental wormwood Content of Chlorogenic Acid, and glycyrrhizic acid is consistent with the test sample preparation method of chlorogenic acid.

(3) method for qualitative and quantitative detection of Qinggan Tablet of the present invention, be a kind of test item more comprehensively, content assaying method is better, and quality control index is more scientific, reasonable, operates the method for quality control guaranteeing the Qinggan Tablet of drug safety and curative effect further more simplified.The method for qualitative and quantitative detection of Qinggan Tablet of the present invention, both may be used for the qualitative and quantitative detection of Qinggan Tablet, can be used for again to other formulations of identical prescription as the qualitative and quantitative detection of the formulation such as capsule, particle.

accompanying drawing explanation:

Fig. 1 is Radix Isatidis microscopical characters figure in Qinggan Tablet of the present invention;

Fig. 2 is the TLC distinguish figure of Radix Isatidis in Qinggan Tablet of the present invention;

Fig. 3 is the TLC distinguish figure of oriental wormwood in Qinggan Tablet of the present invention;

Fig. 4 is the TLC distinguish figure of Radix Glycyrrhizae in Qinggan Tablet of the present invention;

Fig. 5 is glycyrrhizic acid ultraviolet spectrogram;

Fig. 6 is the high-efficient liquid phase chromatogram of glycyrrhizic acid reference substance;

Fig. 7 is the high-efficient liquid phase chromatogram that glycyrrhizic acid content of the present invention measures Qinggan Tablet sample;

Fig. 8 is that Qinggan Tablet of the present invention lacks Radix Glycyrrhizae negative control solution high-efficient liquid phase chromatogram;

Fig. 9 is glycyrrhizic acid linear relationship chart of the present invention;

Figure 10 is chlorogenic acid ultraviolet spectrogram;

Figure 11 is the high-efficient liquid phase chromatogram of chlorogenic acid reference substance;

Figure 12 is the high-efficient liquid phase chromatogram of determination of chlorogenic acid Qinggan Tablet sample of the present invention;

Figure 13 is that Qinggan Tablet of the present invention lacks oriental wormwood negative control solution high-efficient liquid phase chromatogram;

Figure 14 is chlorogenic acid linear relationship chart of the present invention;

Embodiment

embodiment 1

qinggan Tablet: lot number20120502

(1) get this product 5, removing dressing, porphyrize, adds water saturated normal butyl alcohol 5ml, places 0.5 hour, jolting constantly, and filter, filtrate puts evaporate to dryness in water-bath, and residue adds methyl alcohol 1ml and dissolves, as need testing solution; Another extracting Radix Glycyrrhizae control medicinal material 0.5g, is made in the same way of control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 15 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-ethanol-ammonia solution=4:1.5:0.5 for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development in 105 DEG C clear; ; In test sample chromatogram, on the position corresponding to control medicinal material, the spot of aobvious same color;

(3) get this product 5, removing dressing, porphyrize, adds ethanol 15ml, ultrasonic process 45 minutes, and filter, filtrate evaporate to dryness, residue adds ethanol 1ml makes dissolving, as need testing solution; Separately get oriental wormwood control medicinal material 1g, be made in the same way of oriental wormwood control medicinal material solution; Get indigo red reference substance again, add ethanol and make the solution containing 0.05mg in every 1ml; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 15 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=5:0.2:1.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile: 0.01mol/L phosphoric acid solution (30:70) is mobile phase; Flow velocity is 1.0mL/min; Determined wavelength is 250nm; Column temperature 35 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

The preparation of need testing solution: get this product 5, removing dressing, porphyrize, get about 0.2g, put in tool plug conical flask, precision adds 50 ethanol 20ml, weighed weight, ultrasonic process under power 250W, frequency 50kHz condition, let cool, supply the weight of less loss with 50% ethanol, shake up, filter with filter membrane, get subsequent filtrate, to obtain final product;

Assay result: this batch of every sheet of Qinggan Tablet contains Radix Glycyrrhizae with glycyrrhizic acid (C ₄₂h ₆₂o ₁₆) count 0.48mg;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile: 0.01mol/L phosphoric acid solution (5:95) is mobile phase; Flow velocity is 1.0mL/min; Determined wavelength is 327nm; Column temperature 35 DEG C; Number of theoretical plate calculates should be not less than 5000 by chlorogenic acid peak;

The preparation of reference substance solution: get chlorogenic acid reference substance appropriate, put in brown measuring bottle, adds 70% ethanol and makes the solution of every 1ml containing chlorogenic acid 0.01mg; Obtain;

Assay result: this batch of every sheet of Qinggan Tablet this product contains oriental wormwood with chlorogenic acid (C ₁₆h ₁₈o ₉) count 0.17mg.

embodiment 2

qinggan Tablet: lot number20120608

(1) get this product 10, removing dressing, porphyrize, adds water saturated normal butyl alcohol 10ml, places 1 hour, jolting constantly, and filter, filtrate puts evaporate to dryness in water-bath, and residue adds methyl alcohol 2ml and dissolves, as need testing solution; Another extracting Radix Glycyrrhizae control medicinal material 0.3g, is made in the same way of control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-ethanol-ammonia solution=5:2:1 for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development in 105 DEG C clear; ; In test sample chromatogram, on the position corresponding to control medicinal material, the spot of aobvious same color;

(3) get this product 10, removing dressing, porphyrize, adds ethanol 20ml, ultrasonic process 30 minutes, and filter, filtrate evaporate to dryness, residue adds ethanol 2ml makes dissolving, as need testing solution; Separately get oriental wormwood control medicinal material 1.5g, be made in the same way of oriental wormwood control medicinal material solution; Get indigo red reference substance again, add ethanol and make the solution containing 0.1mg in every 1ml; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=7:0.5:2.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile: 0.017mol/L phosphoric acid solution (35:65) is mobile phase; Flow velocity is 0.7mL/min; Determined wavelength is 250nm; Column temperature 30 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

The preparation of need testing solution: get this product 10, removing dressing, porphyrize, get about 0.5g, put in tool plug conical flask, precision adds 70% ethanol 25ml, weighed weight, ultrasonic process under power 250W, frequency 50kHz condition, let cool, supply the weight of less loss with 70% ethanol, shake up, filter with filter membrane, get subsequent filtrate, to obtain final product;

Assay result: this batch of every sheet of Qinggan Tablet contains Radix Glycyrrhizae with glycyrrhizic acid (C ₄₂h ₆₂o ₁₆) count 0.51mg;

Assay result: this batch of every sheet of Qinggan Tablet contains oriental wormwood with chlorogenic acid (C ₁₆h ₁₈o ₉) count 0.19mg.

embodiment 3

qinggan Tablet: lot number20120625

(1) get this product 15, removing dressing, porphyrize, adds water saturated normal butyl alcohol 15ml, places 1.5 hours, jolting constantly, and filter, filtrate puts evaporate to dryness in water-bath, and residue adds methyl alcohol 3ml and dissolves, as need testing solution; Another extracting Radix Glycyrrhizae control medicinal material 1g, is made in the same way of control medicinal material solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with normal butyl alcohol-ethanol-ammonia solution=6:2.5:1.5 for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development in 105 DEG C clear; ; In test sample chromatogram, on the position corresponding to control medicinal material, the spot of aobvious same color;

(3) get this product 15, removing dressing, porphyrize, adds ethanol 50ml, ultrasonic process 60 minutes, and filter, filtrate evaporate to dryness, residue adds ethanol 3ml makes dissolving, as need testing solution; Separately get oriental wormwood control medicinal material 3g, be made in the same way of oriental wormwood control medicinal material solution; Get indigo red reference substance again, add ethanol and make the solution containing 0.2mg in every 1ml; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=9:0.7:3.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile: 0.03mol/L phosphoric acid solution (50:50) is mobile phase; Flow velocity is 0.5mL/min; Determined wavelength is 250nm; Column temperature 25 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

The preparation of need testing solution: get this product 20, removing dressing, porphyrize, get about 1.0g, put in tool plug conical flask, precision adds 80% ethanol 50ml, weighed weight, ultrasonic process under power 250W, frequency 50kHz condition, let cool, supply the weight of less loss with 80% ethanol, shake up, filter with filter membrane, get subsequent filtrate, to obtain final product;

Assay result: this batch of every sheet of Qinggan Tablet contains Radix Glycyrrhizae with glycyrrhizic acid (C ₄₂h ₆₂o ₁₆) count 0.54mg;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile: 0.03mol/L phosphoric acid solution (15:85) is mobile phase; Flow velocity is 0.5mL/min; Determined wavelength is 327nm; Column temperature 25 DEG C; Number of theoretical plate calculates should be not less than 5000 by chlorogenic acid peak;

Assay result: this batch of every sheet of Qinggan Tablet contains oriental wormwood with chlorogenic acid (C ₁₆h ₁₈o ₉) count 0.16mg.

Claims

1. a method for qualitative and quantitative detection for Qinggan Tablet, comprises the Qualitive test of the thin-layered chromatography of Radix Glycyrrhizae, characterized by further comprising and 1. adopts Microscopic Identification method to differentiate Radix Isatidis in Qinggan Tablet; 2. adopt thin-layered chromatography, with oriental wormwood medicinal material for control medicinal material, take indigo red as contrast, differentiate in Qinggan Tablet containing oriental wormwood and Radix Isatidis; 3. the content of high effective liquid chromatography for measuring oriental wormwood Content of Chlorogenic Acid and Radix Glycyrrhizae acid is adopted; Described method for qualitative and quantitative detection comprises following steps:

(3) get this product, removing dressing, porphyrize, adds ethanol, ultrasonic process, and filter, filtrate evaporate to dryness, residue adds ethanol makes dissolving, as need testing solution; Separately get oriental wormwood control medicinal material 1 ~ 3g, be made in the same way of oriental wormwood control medicinal material solution; Get indigo red reference substance again, add ethanol and make solution; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 ~ 15 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=5 ~ 9:0.2 ~ 0.7:1.5 ~ 3.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile-0.01 ~ 0.03mol/L phosphoric acid solution (30 ~ 50:70 ~ 50) is mobile phase; Flow velocity is 0.5-1.0mL/min; Determined wavelength is 250nm; Column temperature 25 ~ 35 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

2. the method for qualitative and quantitative detection of Qinggan Tablet according to claim 1, is characterized in that described method for qualitative and quantitative detection comprises following steps:

(3) get this product 5 ~ 15, removing dressing, porphyrize, adds ethanol 15 ~ 50ml, ultrasonic process 30 ~ 60 minutes, and filter, filtrate evaporate to dryness, residue adds ethanol 1 ~ 3ml makes dissolving, as need testing solution; Separately get oriental wormwood control medicinal material 1 ~ 3g, be made in the same way of oriental wormwood control medicinal material solution; Get indigo red reference substance again, add ethanol and make the solution containing 0.05 ~ 0.2mg in every 1ml; Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 5 ~ 15 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate-acetone=5 ~ 9:0.2 ~ 0.7:1.5 ~ 3.5 for developping agent, launch, take out, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding to indigo red reference substance chromatogram, the spot of aobvious same color; Spray, with 5% potassium hydroxide solution, is inspected under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to oriental wormwood control medicinal material chromatogram, and the fluorescence spot of aobvious same color;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile: 0.01 ~ 0.03mol/L phosphoric acid solution (30 ~ 50:70 ~ 50) is mobile phase; Flow velocity is 0.5 ~ 1.0mL/min; Determined wavelength is 250nm; Column temperature 25 ~ 35 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

3. the method for qualitative and quantitative detection of Qinggan Tablet according to claim 2, is characterized in that described method for qualitative and quantitative detection comprises following steps:

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile-0.017mol/L phosphoric acid solution (35:65) is mobile phase; Flow velocity is 0.7mL/min; Determined wavelength is 250nm; Column temperature 30 DEG C; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;

Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Acetonitrile-0.017mol/L phosphoric acid solution (10:90) is mobile phase; Flow velocity is 0.8mL/min; Determined wavelength is 327nm; Column temperature 30 DEG C; Number of theoretical plate calculates should be not less than 5000 by chlorogenic acid peak;