CN106248860A

CN106248860A - A kind of detection method of ERPIXING KELI

Info

Publication number: CN106248860A
Application number: CN201610887034.6A
Authority: CN
Inventors: 陈修仕; 陈才献
Original assignee: GUIZHOU RUNSHENG PHARMACEUTICAL Co Ltd
Current assignee: GUIZHOU RUNSHENG PHARMACEUTICAL Co Ltd
Priority date: 2016-10-11
Filing date: 2016-10-11
Publication date: 2016-12-21
Anticipated expiration: 2036-10-11
Also published as: CN106248860B

Abstract

The invention discloses the detection method of a kind of ERPIXING KELI, described drug main to be prepared from by Fructus Crataegi, Fructus Hordei Germinatus, Endothelium Corneum Gigeriae Galli, Rhizoma Dioscoreae, Semen Coicis, Semen Lablab Album, Pericarpium Citri Reticulatae and Poria, and described detection method includes discrimination method and content assaying method；Described discrimination method includes that the thin layer to Fructus Crataegi, Pericarpium Citri Reticulatae, Rhizoma Dioscoreae or Endothelium Corneum Gigeriae Galli differentiates detection；Described content assaying method includes the assay to Hesperidin or citric acid.Described detection method is accurate, and highly sensitive, reproducible, testing result is stable, can effectively detect the quality of ERPIXING KELI, and each detection standard can guarantee that curative effect of medication.

Description

A kind of detection method of ERPIXING KELI

Technical field

The present invention relates to the detection method of a kind of ERPIXING KELI, belong to the field of medicine technology；

Background technology

Infantile anorexia is the most common in child, and main symptom has vomiting, inappetence, diarrhoea, constipation, abdominal distention, abdomen Pain and have blood in stool.Children anorexia is the common of department of pediatrics and frequently-occurring disease, if effective prevention can not cause infant hypoevolutism, causes battalion Nourish one's nature anemia, rickets, give infant physical and mental health and cause increasing impact.There is Gastric gysrhythmia in anorexia child simultaneously.

The cause of disease of infantile anorexia is varied, is summed up and mainly has systemic disease, medicine, weather, emotional factor And improper feeding and anorexia nervosa, no matter the infantile anorexia which kind of reason causes, the persistent period is longer all can affect little Youngster's height, the normal growth of body weight, merging is become thin, malnutrition, hypoimmunity, the most then develop into infantile malnutrition sick, show as yellowish complexion Leanness, hypotrichosis, abdominal flatulence blue veins expose or abdomen is recessed such as boat etc., have a strong impact on the growth promoter of children's, and easily initiation is also Send out disease.Western medicine because of often cause allergy, feel sick, the toxic and side effects such as diarrhoea, liver function injury is the most increasingly avoided for people as far as possible, Treatment infantile anorexia first-selection Chinese medicine, when the traditional Chinese medical science tackles this type of disease, often can use effect substantially and untoward reaction is less Medicine.Medicine tool with the ERPIXING KELI that Fructus Crataegi, Fructus Hordei Germinatus, Endothelium Corneum Gigeriae Galli, Rhizoma Dioscoreae, Semen Coicis, Semen Lablab Album, Pericarpium Citri Reticulatae and Poria are made There is invigorating the spleen and regulating the stomach, effect of promoting digestion and removing stagnation.Can be used for the infantile anorexia that insufficiency of the spleen dyspepsia causes, loose stool, weak disease such as grade of becoming thin. In order to better control over the quality of this product, it is ensured that clinical drug effect, the invention provides the detection method of this medicine.

Summary of the invention

It is an object of the invention to, it is provided that the detection method of a kind of ERPIXING KELI；Described detection method is accurate, sensitivity Height, reproducible, reliable results, can effectively control the quality of this product, it is ensured that the clinical drug effect of medicine.

For solving above-mentioned technical problem, the present invention adopts the following technical scheme that realization:

The detection method of a kind of ERPIXING KELI, described granule is mainly by the Fructus Crataegi 300-340 part calculated by weight, wheat Bud

300-340 part, Endothelium Corneum Gigeriae Galli 220-260 part, Rhizoma Dioscoreae 220-260 part, Semen Coicis 140-180 part, Semen Lablab Album 220-260 Part, Pericarpium Citri Reticulatae 86-106 part and Poria 86-106 part are made as follows: above eight tastes, and Fructus Crataegi, tangerine peel powder are broken into coarse powder, use 65% ethanol as solvent, after impregnating 24 hours, carries out percolation, collects liquid of filtering, diacolation liquid recycling ethanol, be concentrated into 55-65 DEG C of phase It is the thick paste of 1.35-1.40 to density, standby；The Six-element medical material boilings such as remaining Fructus Hordei Germinatus three times, each 1 hour, merge and decoct Liquid, filters, and when filtrate is concentrated into 55-65 DEG C, relative density is the clear paste of 1.10, adds equivalent ethanol and makes precipitation, takes supernatant, returns Receive ethanol, be concentrated into the thick paste that relative density is 1.35-1.40；With above-mentioned Fructus Crataegi, the mixing of Pericarpium Citri Reticulatae thick paste, add sucrose in right amount, Make granule, be dried, to obtain final product；Described detection method includes discrimination method and content assaying method；It is right that described discrimination method includes The thin layer of Fructus Crataegi, Pericarpium Citri Reticulatae, Rhizoma Dioscoreae or Endothelium Corneum Gigeriae Galli differentiates detection；Described content assaying method includes containing Hesperidin or citric acid It is fixed to measure.

The detection method of aforesaid ERPIXING KELI, the discrimination method of described Fructus Crataegi is: composition granule of getting it filled, finely ground after, take 4-12g adds 20-30ml ether, is heated to reflux 0.5-1.5 hour, filters, and filtrate volatilizes, and residue adds methanol 0.5-1.5m1 to be made molten Solve, as need testing solution；Separately take Fructus Crataegi control medicinal material 0.1-0.3g, finely ground after, take 4-12g and add 20-30ml ether, heat back Flowing 0.5-1.5 hour, filter, filtrate volatilizes, and residue adds methanol 0.5-1.5m1 makes dissolving, prepares control medicinal material solution；Take Bears again Fruit acid reference substance is appropriate, adds methanol and makes every 0.5-1.5ml solution containing 0.05-0.15mg, as reference substance solution；According to State's pharmacopeia thin layer chromatography test, draws above-mentioned three all solution each 5-10 μ l, puts respectively on same silica gel g thin-layer plate, with Toluene: ethyl acetate: formic acid=15-25: 2-6: 0.1-1.0 is developing solvent, launches, and takes out, dries, spray molten with sulphuric acid ethanol Liquid, in ethanol solution of sulfuric acid, sulphuric acid is 2-4:9-11 with the volume ratio of ethanol；It is heated to spot development clear, respectively at 105 DEG C Put and inspect under daylight or 360-370nm ultra-violet lamp, in test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious The principal spot of same color or fluorescence principal spot；On position corresponding with reference substance chromatograph, the speckle or glimmering of aobvious same color Light speckle.

The detection method of aforesaid ERPIXING KELI, the discrimination method of described Fructus Crataegi is: composition granule of getting it filled, finely ground after, take 8g adds 25ml ether, is heated to reflux 1 hour, filters, and filtrate volatilizes, and residue adds methanol 1m1 makes dissolving, as need testing solution； Separately take Fructus Crataegi control medicinal material 0.2g, finely ground after, take 8g and add 25ml ether, be heated to reflux 1 hour, filter, filtrate volatilizes, and residue adds Methanol 1m1 makes dissolving, prepares control medicinal material solution；Take ursolic acid reference substance more appropriate, add methanol and make every 1ml containing 0.1mg's Solution, as reference substance solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned three all solution each 5～10 μ l, point Other point is on same silica gel g thin-layer plate, with toluene: ethyl acetate: formic acid=20: 4: 0.5 as developing solvent, launches, and takes out, dries in the air Dry, spray with ethanol solution of sulfuric acid, in ethanol solution of sulfuric acid, sulphuric acid is 3:10 with the volume ratio of ethanol；It is heated to speckle at 105 DEG C Colour developing is clear, puts respectively and inspects under daylight or 365nm ultra-violet lamp, in test sample chromatograph, corresponding with control medicinal material chromatograph On position, the principal spot of aobvious same color or fluorescence principal spot；On position corresponding with reference substance chromatograph, aobvious same color Speckle or fluorescence speckle.

The detection method of aforesaid ERPIXING KELI, the discrimination method of described Pericarpium Citri Reticulatae is: composition granule of getting it filled, finely ground after, take 5-15g, adds methanol 25-35ml, is heated to reflux 0.5-1.5 hour, filters, and filtrate volatilizes, and the residue 5-15ml that adds water makes dissolving, turns Moving in separatory funnel, extract 2-3 time with ethyl acetate shaking, each each 5-15ml, combined ethyl acetate extracting solution, in water-bath Being evaporated, residue adds methanol 1-4ml makes dissolving, as need testing solution；Separately taking Pericarpium Citri Reticulatae control medicinal material 0.5-1.5g, it is right to be made in the same way of According to medical material solution；Take Hesperidin reference substance more appropriate, add methanol and make saturated solution, as reference substance solution；According to middle traditional Chinese medicines Allusion quotation thin layer chromatography is tested, and draws above-mentioned three kinds of solution each 2-5 μ l, puts respectively in same 0.1-1.0% sodium hydroxide solution On the silica gel g thin-layer plate of preparation, with ethyl acetate: methanol: water=95-105: 15-19: 10-16, as developing solvent, opens up to 1-6cm, Take out, dry, then with toluene: ethyl acetate: formic acid: the upper solution of water=15-25: 5-15: 0.5-1.5: 0.5-1.5 is for exhibition Opening agent, open up to 5-10cm, take out, dry, spray is with 1-10% aluminum chloride ethanol solution, and being placed in wavelength is 360-370nm ultraviolet Inspect under light modulation；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the fluorescence principal spot of aobvious same color； On position corresponding with reference substance chromatograph, the fluorescence speckle of aobvious same color.

The detection method of aforesaid ERPIXING KELI, the discrimination method of described Endothelium Corneum Gigeriae Galli is: composition granule of getting it filled, finely ground, takes 1-10g, add water 25-35ml, steeped overnight, and aqueous solution, together with in residue dislocation separatory funnel, adds water saturated n-butyl alcohol Shaking is extracted 2-4 time, each 15-25ml, divides and takes n-butyl alcohol liquid, is evaporated, and residue adds proper amount of methanol makes dissolving, is added on 100-120 Mesh, 1-10g, internal diameter 10-15mm neutral alumina column on, with 5-15% methanol 90-110ml eluting, collect eluent, steam Dry, residue adds 0.5-1.5ml, 65-75% ethanol makes dissolving, as need testing solution；Separately take Endothelium Corneum Gigeriae Galli control medicinal material 0.5- 1.5g, add water 25-35ml, decocts 1-3 hour, and aqueous solution, together with in residue dislocation separatory funnel, is made in the same way of comparison medicine Material solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above two solution each 3～5 μ l, put respectively in same silica gel G On lamellae, with n-butyl alcohol: glacial acetic acid: water=5-15: 1-6: the 0.5-1.5 lamellae 10-as developing solvent, after pre-equilibration point sample 20 minutes, launching, take out, dry, spray, with ninhydrin solution, is heated to spot development at 110 DEG C clear；In test sample chromatograph, On position corresponding with control medicinal material chromatograph, the principal spot of aobvious same color.

The detection method of aforesaid ERPIXING KELI, the discrimination method of described Endothelium Corneum Gigeriae Galli is: composition granule of getting it filled, finely ground, takes 5g, add water 30ml, steeped overnight, and aqueous solution, together with in residue dislocation separatory funnel, adds the shaking of water saturated n-butyl alcohol and carries Taking 3 times, each 20ml, divide and take n-butyl alcohol liquid, be evaporated, residue adds proper amount of methanol makes dissolving, is added on 100-120 mesh, 5g, internal diameter On the neutral alumina column of 10-15mm, with 10% methanol 100ml eluting, collecting eluent, be evaporated, residue adds 1ml70% ethanol Make dissolving, as need testing solution；Separately taking Endothelium Corneum Gigeriae Galli control medicinal material 1g, add water 30ml, decocts 2 hours, and aqueous solution is together with residue Together in dislocation separatory funnel, it is made in the same way of control medicinal material solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned two Plant solution each 3～5 μ l, put respectively on same silica gel g thin-layer plate, with n-butyl alcohol: glacial acetic acid: water=9: 3: 1 as developing solvent, in advance Lamellae after balance point sample 15 minutes, launches, and takes out, dries, and spray, with ninhydrin solution, is heated to spot development at 110 DEG C Clearly；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the principal spot of aobvious same color.

The detection method of aforesaid ERPIXING KELI, described content of hesperidin assay method is:

According to Chinese Pharmacopoeia high effective liquid chromatography for measuring:

Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；With methanol: ice vinegar Acid: water=30-40: 1-8: 55-65 is flowing phase；Column temperature is 33-37 DEG C；Detection wavelength is 280-286nm；Number of theoretical plate is by orange Skin glycosides peak calculates should be not less than 3000；

The preparation of reference substance solution: take Hesperidin reference substance appropriate, accurately weighed, add methanol and make every 0.5-1.5ml and contain The solution of 0.005-0.015mg, to obtain final product；

The preparation of need testing solution: composition granule content of getting it filled, mixing, finely ground, take 0.2-0.6g, accurately weighed, put tool In plug conical flask, accurate addition methanol 20-30ml, close plug, weighed weight, supersound process, sonification power is 200-300W, Supersound process frequency is 30-40kHz, and sonication treatment time is 25-35 minute, lets cool, more weighed weight, supplies less loss with methanol Weight, shake up, filter, take subsequent filtrate, to obtain final product；

Algoscopy: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, Obtain.

Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；With methanol: ice vinegar Acid: water=35: be flowing phase at 4: 61；Column temperature is 35 DEG C；Detection wavelength is 283nm；Number of theoretical plate presses the calculating of Hesperidin peak should not Less than 3000；

The preparation of reference substance solution: take Hesperidin reference substance appropriate, accurately weighed, add methanol and make every 1ml containing 0.01mg Solution, to obtain final product；

The preparation of need testing solution: composition granule content of getting it filled, mixing, finely ground, take 0.4g, accurately weighed, put tool plug cone In shape bottle, accurate addition methanol 25ml, close plug, weighed weight, supersound process, sonification power is 250W, supersound process frequency Rate is 35kHz, and sonication treatment time is 30 minutes, lets cool, more weighed weight, supplies the weight of less loss with methanol, shakes up, filter Cross, take subsequent filtrate, to obtain final product；

The detection method of aforesaid ERPIXING KELI, described citric acid content assaying method is: efficient according to Chinese Pharmacopoeia Liquid chromatography for measuring:

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；Number of theoretical plate presses Chinese holly Rafter acid peak calculates should be not less than 3000；

The preparation of reference substance solution: take citric acid reference substance appropriate, accurately weighed, add water and make every 0.5-1.5ml and contain The solution of 0.1-0.6mg, to obtain final product；

The preparation of need testing solution: composition granule content of getting it filled, finely ground, precision weighs 1-4g, puts in 20-30ml measuring bottle, The accurate water that adds is appropriate, and supersound process, sonification power is 200-300W, and supersound process frequency is 30-40kHz, ultrasonic place The reason time is 15-25 minute, lets cool, and is diluted with water to scale and shakes up, and filters, and takes subsequent filtrate, crosses the filter of 0.35-0.55 μm micropore Film, to obtain final product.

Algoscopy: precision draws reference substance solution and need testing solution each 15-25 μ l respectively, injects chromatograph of liquid, surveys Fixed, to obtain final product.

The detection method of aforesaid ERPIXING KELI, the discrimination method of described Fructus Crataegi is: after composition granule of getting it filled is finely ground, take 8g Adding 25ml ether, be heated to reflux 1 hour, filter, filtrate volatilizes, and residue adds methanol 1m1 makes dissolving, as need testing solution；Separately Take Fructus Crataegi control medicinal material 0.2g, finely ground after, take 8g and add 25ml ether, be heated to reflux 1 hour, filter, filtrate volatilizes, and residue adds first Alcohol 1m1 makes dissolving, prepares control medicinal material solution；Take ursolic acid reference substance more appropriate, add methanol and make molten containing 0.1mg of every 1ml Liquid, as reference substance solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned three all solution each 5～10 μ l, respectively Point is on same silica gel g thin-layer plate, with toluene: ethyl acetate: formic acid=20: 4: 0.5 as developing solvent, launches, takes out, dry, Spraying with ethanol solution of sulfuric acid, in ethanol solution of sulfuric acid, sulphuric acid is 3:10 with the volume ratio of ethanol；It is heated to spot development at 105 DEG C Clearly, put respectively and inspect under daylight or 365nm ultra-violet lamp, in test sample chromatograph, in position corresponding with control medicinal material chromatograph On, the principal spot of aobvious same color or fluorescence principal spot；On position corresponding with reference substance chromatograph, the speckle of aobvious same color Or fluorescence speckle；

The discrimination method of described Pericarpium Citri Reticulatae is: composition granule content of getting it filled, finely ground after, take 10g, add methanol 30ml, heat back Flowing 1 hour, filter, filtrate volatilizes, and the residue 10ml that adds water makes dissolving, is transferred in separatory funnel, shakes extraction 2 by ethyl acetate Secondary, each each 10ml, combined ethyl acetate extracting solution, water-bath is evaporated, residue adds methanol 2ml makes dissolving, molten as test sample Liquid；Separately take Pericarpium Citri Reticulatae control medicinal material 1g, be made in the same way of control medicinal material solution；Take Hesperidin reference substance more appropriate, add methanol and make full And solution, as reference substance solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned three kinds of solution each 2～5 μ l, point Other point is on the same silica gel g thin-layer plate prepared with 0.5% sodium hydroxide solution, with ethyl acetate: methanol: water=100: 17: 13 is developing solvent, opens up to about 3cm, takes out, dry, then with toluene: ethyl acetate: formic acid: water=20: the upper solution of 10: 1: 1 For developing solvent, opening up to 8cm, take out, dry, spray is with 5% aluminum chloride ethanol solution, and being placed in wavelength is under 365nm ultra-violet lamp Inspect；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the fluorescence principal spot of aobvious same color；With right According on the corresponding position of product chromatograph, show the fluorescence speckle of same color；

The discrimination method of described Endothelium Corneum Gigeriae Galli is: composition granule of getting it filled, finely ground, takes 5g, and add water 30ml, steeped overnight, aqueous solution Together with in residue dislocation separatory funnel, add the shaking of water saturated n-butyl alcohol and extract 3 times, each 20ml, divide and take n-butyl alcohol liquid, Being evaporated, residue adds proper amount of methanol makes dissolving, is added on the neutral alumina column of 100-120 mesh, 5g, internal diameter 10-15mm, with 10% Methanol 100ml eluting, collects eluent, is evaporated, and residue adds 1ml70% ethanol makes dissolving, as need testing solution；Separately take in chicken Gold control medicinal material 1g, add water 30ml, decocts 2 hours, and aqueous solution, together with in residue dislocation separatory funnel, is made in the same way of comparison Medical material solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above two solution each 3～5 μ l, put respectively in same silica gel On G lamellae, with n-butyl alcohol: glacial acetic acid: water=9: 3: 1 as developing solvent, the lamellae after pre-equilibration point sample 15 minutes, launch, Taking out, dry, spray, with ninhydrin solution, is heated to spot development at 110 DEG C clear；In test sample chromatograph, with control medicinal material On the corresponding position of chromatograph, the principal spot of aobvious same color；

Described content of hesperidin assay method is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring:

Algoscopy: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, Obtain；Described citric acid content assaying method is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring:

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With 5% biphosphate Ammonium salt solution, is flowing phase with phosphoric acid slow-readjustment joint pH to 3.0, and column temperature is 25 DEG C；Detection wavelength is 210nm；Number of theoretical plate presses citron Acid peak calculates and should be not less than 3000；

The preparation of reference substance solution: take citric acid reference substance appropriate, accurately weighed, add water and make molten containing 0.3mg of every 1ml Liquid, to obtain final product；

The preparation of need testing solution: composition granule content of getting it filled, finely ground, precision weighs 2.0g, puts in 25ml measuring bottle, accurate Adding water appropriate, supersound process, sonification power is 250W, and supersound process frequency is 35kHz, and sonication treatment time is 20 points Clock, lets cool, and is diluted with water to scale and shakes up, and filters, and takes subsequent filtrate, crosses 0.45 μm microporous filter membrane, to obtain final product.

Algoscopy: precision draws reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid, measures, Obtain.

Inventor has carried out substantial amounts of experiment, the following is the research of detection method of the present invention

Experimental example: detection method research

One, sample

Sample: ERPIXING KELI, is prepared as described in Example 1, for the granule of brown color；Feeble QI is fragrant, sweet and sour, Micro-hardship.Recording in WS-10441 (ZD-0441)-2002-2012Z, in standard, original project is: character；Fructus Crataegi, Pericarpium Citri Reticulatae thin layer mirror Not；Check；Assay project is the mensuration of Hesperidin in Pericarpium Citri Reticulatae；According to improving quality criteria requirements, intend primary standard project Carry out revising and Rhizoma Dioscoreae, the discriminating of Endothelium Corneum Gigeriae Galli thin layer, Fructus Crataegi assay in the side of adding.Through experimentation to this product quality standard Propose to increase revision project and carried out Method validation, increase revision checking Description Experimental according to as follows.

Two, differentiate

1, the thin layer of Fructus Crataegi differentiates:

Primary standard is recorded and is only used ursolic acid as comparison in method, and specificity is strong, therefore here, is repaiied this discriminating Order, add Fructus Crataegi comparison material.Revision method is: take the content under this product content uniformity item, finely ground, takes about 8g, adds diethyl ether 25ml, is heated to reflux 1 hour, filters, and filtrate volatilizes, and residue adds methanol 1m1 makes dissolving, as need testing solution.Separately take Fructus Crataegi Control medicinal material 0.2g, obtains control medicinal material solution with legal system.Take ursolic acid reference substance more appropriate, add methanol and make every 1ml containing 0.1mg Solution, as reference substance solution.Test according to thin layer chromatography (Chinese Pharmacopoeia four general rules 0502 of version in 2015), draw above-mentioned Three all solution each 5～10 μ l, put on same silica gel g thin-layer plate, respectively with toluene-ethyl acetate-formic acid (20: 4: 0.5) For developing solvent, launching, take out, dry, spray, with ethanol solution of sulfuric acid (3 → 10), is heated to spot development at 105 DEG C clear.Point Do not put and inspect under daylight or ultra-violet lamp (365nm), in test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious The principal spot of same color or fluorescence principal spot；On position corresponding with reference substance chromatograph, the speckle or glimmering of aobvious same color Light speckle.On request, this discriminating is carried out methodological study, through Method validation, this thin-layer identification method clear spot, Separator well, reappears, and negative noiseless.Method validation is as follows:

1.1 reagent and reagent

ERPIXING KELI is provided by Guizhou Run Sheng pharmacy industry company limited, is i.e. prepared by embodiment 1；Fructus Crataegi comparison medicine Material (121626-201402), ursolic acid reference substance (110742-201421) are provided by Nat'l Pharmaceutical & Biological Products Control Institute； Silica gel g thin-layer plate: the silica gel g thin-layer plate of subsidiary factory of Haiyang Chemical Plant, Qingdao production and self-control silica gel g thin-layer plate；Reagent: toluene, second Acetoacetic ester, formic acid are analytical pure；

1.2 specificity

Take ERPIXING KELI, Fructus Crataegi control medicinal material, ursolic acid reference substance and scarce Fructus Crataegi negative sample, with method described in text Prepare need testing solution, control medicinal material solution, reference substance solution and negative need testing solution, respectively point sample, launch, develop the color, put Daylight or ultra-violet lamp (365nm), in test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious same color Principal spot or fluorescence principal spot；On position corresponding with reference substance chromatograph, the speckle of aobvious same color or fluorescence speckle.And speckle Point separates preferably, negative noiseless, is specifically shown in Fig. 1 and Fig. 2.

2, Pericarpium Citri Reticulatae thin layer differentiates

After recording method extraction point sample due to primary standard, sample impurity is more, has certain interference, differentiates to extract to this at this The further remove impurity of method, adds reference substance point sample simultaneously, and method is defined as: takes the content under this product content uniformity item, grinds Carefully, taking about 10g, add methanol 30ml, be heated to reflux 1 hour, filter, filtrate volatilizes, and the residue 10ml that adds water makes dissolving, is transferred to point In liquid funnel, extract 2 times with ethyl acetate shaking, each each 10ml, combined ethyl acetate extracting solution, water-bath is evaporated, residue Add methanol 2ml and make dissolving, as need testing solution.Separately take Pericarpium Citri Reticulatae control medicinal material 1g, be made in the same way of control medicinal material solution.Take orange again Skin glycosides reference substance is appropriate, adds methanol and makes saturated solution, as reference substance solution.According to thin layer chromatography (Chinese Pharmacopoeia 2015 Four general rules 0502 of version) test, draw above-mentioned three kinds of solution each 2～5 μ l, put respectively and use 0.5% sodium hydroxide solution in same On the silica gel g thin-layer plate of preparation, with acetate-methanol-water 100: 17: 13) as developing solvent, open up to about 3cm, take out, dry, Again with the upper solution of toluene-ethyl acetate-formic acid-water (20: 10: 1: 1) as developing solvent, open up to about 8cm, take out, dry, spray With 5% aluminum chloride ethanol solution, put and inspect under ultra-violet lamp (365nm).In test sample chromatograph, with control medicinal material chromatograph phase On the position answered, the fluorescence principal spot of aobvious same color；On position corresponding with reference substance chromatograph, the fluorescence of aobvious same color Speckle.This discriminating has been carried out methodological study, through Method validation, this thin-layer identification method clear spot, separator well, weight The best and negative noiseless.Method validation is as follows:

2.1 reagent and reagent

ERPIXING KELI is provided by Guizhou Run Sheng pharmacy industry company limited；Pericarpium Citri Reticulatae control medicinal material (120969-201109) and Hesperidin reference substance (110721-201014) is provided by Nat'l Pharmaceutical & Biological Products Control Institute；Lamellae: Qingdao Haiyang chemical industry The silica gel g thin-layer plate that subsidiary factory of factory produces immerses 0.5% sodium hydroxide solution, takes out, dries；Make 0.5% sodium hydroxide solution by oneself The silica gel g thin-layer plate of preparation；Reagent: toluene, ethyl acetate, formic acid, methanol are analytical pure；

2.2, specificity

Take ERPIXING KELI, Pericarpium Citri Reticulatae control medicinal material, Hesperidin reference substance and scarce Pericarpium Citri Reticulatae negative sample, with method described in text Prepare need testing solution, control medicinal material solution, reference substance solution and negative need testing solution, respectively point sample, launch, develop the color, put Inspecting under ultra-violet lamp (365nm), in test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious same color is glimmering Light principal spot, on position corresponding with reference substance chromatograph, the fluorescence speckle of aobvious same color.And speckle separates preferably, negative Noiseless, see Fig. 3.

2.3 Rhizoma Dioscoreae thin layers differentiate

Through overtesting, method is defined as: composition granule of getting it filled, finely ground, takes about 5g, and add methylene chloride 50ml, is heated to reflux 2 little Time, filtering, take filtrate and put in separatory funnel, wash with water 3 times, each 50ml, divide and take dichloromethane solution, be evaporated, residue adds 1ml Dichloromethane makes dissolving, as need testing solution.Separately take Rhizoma Dioscoreae control medicinal material 0.5g, be made in the same way of control medicinal material solution.According to thin Layer chromatography (Chinese Pharmacopoeia four general rules 0502 of version in 2015) test, draw above two solution each 5～10 μ l, put respectively in Lamellae 15 on same silica gel g thin-layer plate, with chloroform-methanol-water (16: 3: 0.1) as developing solvent, after pre-equilibration point sample Minute, launching, take out, dry, spray, with phosphomolybdic acid test solution, is heated to spot development at 110 DEG C clear.In test sample chromatograph, On position corresponding with control medicinal material chromatograph, the principal spot of aobvious same color.By pharmacopoeial requirements, this discriminating is carried out methodology Investigate, through Method validation, this thin-layer identification method clear spot, separator well, reappear, and negative noiseless.Methodology Verify as follows:

2.3.1, reagent and reagent

ERPIXING KELI Guizhou Run Sheng pharmacy industry company limited provides；Rhizoma Dioscoreae control medicinal material (121137-200402) by State's medicine biological products assay institute provides；Silica gel thin-layer plate: the silica gel g thin-layer plate of subsidiary factory of Haiyang Chemical Plant, Qingdao production and self-control Silica gel g thin-layer plate reagent: dichloromethane, chloroform, methanol are analytical pure, water is super purified water；

2.3.2, specificity

Take ERPIXING KELI, Rhizoma Dioscoreae control medicinal material and scarce Rhizoma Dioscoreae negative sample, prepare test sample with method described in text molten Liquid, control medicinal material solution, reference substance solution and negative need testing solution, respectively point sample, launch, spray with phosphomolybdic acid test solution, 110 DEG C it is heated to spot development clear, in test sample chromatograph, the master of aobvious same color on position corresponding with control medicinal material chromatograph Speckle, and speckle separation is preferably, negative noiseless, result is shown in Fig. 4.

The thin layer of 2.4 Endothelium Corneum Gigeriae Galli differentiates

Through overtesting, method is defined as: composition granule of getting it filled, finely ground, takes about 5g, and add water 30ml, steeped overnight, and aqueous solution is even With in residue together dislocation separatory funnel, add the shaking of water saturated n-butyl alcohol and extract 3 times, each 20ml, divides and takes n-butyl alcohol liquid, steam Dry, residue adds proper amount of methanol makes dissolving, is added on neutral alumina column (100-120 mesh, 5g, internal diameter 10-15mm), uses 10% first Alcohol 100ml eluting, collects eluent, is evaporated, and residue adds 1ml70% ethanol makes dissolving, as need testing solution.Separately take Endothelium Corneum Gigeriae Galli Control medicinal material 1g, add water 30ml, decocts 2 hours, and aqueous solution, together with in residue dislocation separatory funnel, is made in the same way of comparison medicine Material solution.Test according to thin layer chromatography (Chinese Pharmacopoeia four general rules 0502 of version in 2015), draw above two solution each 3～5 μ L, puts respectively on same silica gel g thin-layer plate, with n-butyl alcohol-glacial acetic acid-water (9:3:1) as developing solvent, after pre-equilibration point sample Lamellae 15 minutes, launches, and takes out, dries, and spray, with ninhydrin solution, is heated to spot development at 110 DEG C clear.Test sample color In spectrum, on position corresponding with control medicinal material chromatograph, the speckle of aobvious same color.By pharmacopoeial requirements, this discriminating is carried out Methodological study, through Method validation, this thin-layer identification method clear spot, separator well, reappear, and negative noiseless. Method validation is as follows:

2.4.1 reagent and reagent

ERPIXING KELI is provided by Guizhou Run Sheng pharmacy industry company limited；Endothelium Corneum Gigeriae Galli control medicinal material (1153-200001) by Nat'l Pharmaceutical & Biological Products Control Institute provides；Silica gel g thin-layer plate: subsidiary factory of Haiyang Chemical Plant, Qingdao produce silica gel g thin-layer plate and Self-control silica gel g thin-layer plate；Reagent: methanol, ethanol, n-butyl alcohol are analytical pure, water is super purified water；

2.4.2 specificity

Take ERPIXING KELI, Endothelium Corneum Gigeriae Galli control medicinal material and scarce Endothelium Corneum Gigeriae Galli negative sample, prepare for examination with method described in text Product solution, control medicinal material solution, reference substance solution and negative need testing solution, respectively point sample, launch, spray with ninhydrin solution, It is heated to spot development clear at 110 DEG C, in test sample chromatograph, aobvious same color on position corresponding with control medicinal material chromatograph Principal spot, and speckle separates preferably, negative noiseless, sees Fig. 5.

Two, assay

1 content of hesperidin measures

Pericarpium Citri Reticulatae: Pericarpium Citri Reticulatae assay, for record for primary standard, there is no revision, as requested, records primary standard old at this Skin assay has carried out Method validation, and Method validation is as follows:

1.1 instrument and reagent

High performance liquid chromatograph: Shimadzu chromatograph of liquid；ERPIXING KELI: Guizhou Runsheng Pharmaceutical Co., Ltd.；Hesperidin Reference substance is provided by Nat'l Pharmaceutical & Biological Products Control Institute, and lot number is 110721-201014；Reagent: methanol is that chromatograph alcohol, water are Double distilled water.

1.2 assay method

1.2.1 chromatography condition:

(1) chromatographic column: with reference to primary standard and version pharmacopeia Pericarpium Citri Reticulatae assay in 2010, selects octadecylsilane bonded silica Glue is that the pillar of filler is investigated.Result shows that the pillar using octadecylsilane chemically bonded silica to be filler separates effect Fruit is preferably.

(2) flowing phase: with reference to primary standard, uses methanol-glacial acetic acid-water (35: 4: 61) for flowing phase, composition Pericarpium Citri junoris to be measured Glycosides and the available preferably separation of other impurity.

(3) detection wavelength: measuring with reference to primary standard and version pharmacopeia content of hesperidin in 2010, detection wavelength is set to 283nm.

(4) theoretical cam curve: according to different pillar testing results, referring concurrently to version pharmacopeia Pericarpium Citri Reticulatae assay in 2010, Tentative theoretical cam curve is pressed Hesperidin peak and is calculated, and should be not less than 3000.

1.2.2 the preparation of need testing solution

Get it filled composition granule, mixing, finely ground, take about 0.4g, accurately weighed, put in tool plug conical flask, accurate add methanol 25ml, close plug, weighed weight, supersound process (power 250W, frequency 35kHz) 30 minutes, let cool, more weighed weight, use methanol Supply less loss weight, shake up, filter, take subsequent filtrate, to obtain final product.

1.2.3, the preparation of reference substance solution

Take Hesperidin reference substance appropriate, accurately weighed, add methanol and make every 1ml solution containing 0.01mg, to obtain final product.

1.3 method validation

The most linearly investigate

Precision absorption Hesperidin reference substance solution (0.031906mg/ml) 1ml, 2ml, 3ml, 5ml, 6ml, 10ml arrive respectively In 10ml volumetric flask, add methanol to scale, point take 10 μ l sample introductions, record chromatograph, with peak area as vertical coordinate, with sample size (μ g) Mapping for abscissa, calculating regression equation is: A=1847322.47C+3897.83801 (r=0.9999), regression equation matching The linear equation crossing initial point is: A=1866179.663C；See that Fig. 6 and Biao 1. is by a need testing solution sample introduction analysis, face, gained peak Integration does not substitute into two formulas and calculates, and relative deviation is 0.32%, therefore it is believed that standard curve crosses initial point, regression equation intercept is Zero.Thus assay can use one point external standard method to calculate.Hesperidin linear relationship between 0.032 μ g～0.319 μ g is good.

Table 1 Hesperidin linear relationship is investigated

1.3.2, precision test

Take reference substance solution 0.011867mg/ml, repeat sample introduction 6 times, record chromatograph, the results are shown in Table 2 and precision collection of illustrative plates, RSD is 0.41%, and good precision is described.

Table 2 precision test

1.3.3, replica test

Take a sample as described in text, prepare 6 parts of need testing solutions, respectively sample introduction, record chromatograph, calculate, the results are shown in Table 3, Repeatability collection of illustrative plates, RSD is 0.58%, and good repeatability is described.

Table 3 replica test

1.3.4, recovery test

Using sample-adding to reclaim, accurate absorption has measured the sample 0.2g (0.5789mg/g) of content, adds Hesperidin pair Appropriate according to product, as described in text, chromatographic condition measures, and calculates the response rate, the results are shown in Table 4, response rate collection of illustrative plates, the recovery of Hesperidin Rate is 97.81%, and RSD is 0.75%, illustrates that this law has the good response rate.

Table 4 recovery test

1.3.5, specificity

Take ERPIXING KELI and negative sample and prepare sample solution and negative sample solution by need testing solution preparation method, By above-mentioned chromatographic condition, take reference substance solution, sample solution, negative sample solution injection chromatograph of liquid, sample solution respectively Chromatograph is equipped with corresponding chromatographic peak in reference substance solution chromatograph corresponding positions, and negative noiseless, and result is shown in Fig. 8, Fig. 9 and Figure 10.

1.3.6, ruggedness

1.3.6.1 the selection of sample extraction solvent

Get it filled composition granule, finely ground, take 5 parts, every part of about 0.4g, accurately weighed, put in tool plug conical flask, precision adds respectively Ethanol, methanol, 60% methanol, 70% methanol, the 80% each 25ml of methanol, weighed weight, supersound process 30 minutes, let cool, then claim Determine weight, supply the weight of less loss respectively with ethanol, methanol, 60% methanol, 70% methanol, 80% methanol, shake up, filter, take Subsequent filtrate is as need testing solution.Take each 10 μ l of above-mentioned solution respectively and inject chromatograph of liquid, calculate, the results are shown in Table 5, this is described Product use methanol to extract more complete as Extraction solvent, content.

The selection of table 5 Extraction solvent

1.3.6.2, the selection of sample extraction method

Get it filled composition granule, finely ground, take 2 parts, every part of about 0.4g, accurately weighed, to put in tool plug conical flask for the 1st part, precision adds Enter methanol 25ml, supersound process 30 minutes, another part of reflux, extract, 30 minutes, take out, let cool, supply less loss weight with methanol, Shake up, filter；Take above-mentioned 2 kinds of solution 10 μ l respectively and inject chromatograph of liquid, calculate, the results are shown in Table 6, reflux, extract, content, super Sound extraction content is without essential difference, therefore selects supersound extraction.

The selection of table 6 extracting method

1.3.6.3, sample extraction time effects

Take the content drug particles under content uniformity item, finely ground, take 3 parts, every part of about 0.4g, accurately weighed, put respectively In tool plug conical flask, the accurate methanol 25ml that adds, weighed weight, respectively supersound extraction 10,15,30,40 minutes, take out, let cool, Supply less loss weight with methanol, shake up, filter, take 10 μ l subsequent filtrates respectively and inject chromatograph of liquid, calculate content, the results are shown in Table 7, supersound extraction is extracted for 30 minutes the most completely, therefore the selective extraction time is 30 minutes.

Table 7 extraction time affects

Extracting method is defined as: composition granule of getting it filled, mixing, finely ground, takes about 0.4g, accurately weighed, puts in tool plug conical flask, Accurate addition methanol 25ml, close plug, weighed weight, supersound process (power 250W, frequency 35kHz) 30 minutes, let cool, more weighed Weight, supplies less loss weight with methanol, shakes up, and filters, takes subsequent filtrate, to obtain final product.

1.3.6.4, the impact of different brands pillar

Using different brands octadecylsilane chemically bonded silica is the pillar of filler, determines respectively in ERPIXING KELI The content of Hesperidin, investigates the impact of the chromatographic column of different brands.Assay the results are shown in Table 8.Result shows different brands ten Eight alkyl silane bonded silica gels be filler chromatographic column on assay without impact.

The measurement result of table 8 different brands chromatographic column

1.3.6.5, sample stability test

Take ERPIXING KELI, prepare by need testing solution preparation method, measure Hesperidin peak area by above-mentioned chromatographic condition, The results are shown in Table 9.

Table 9 sample stability is tested

1.3.7 ten batches of content sample determinations: result table 10；

Table 10 10 batch sample assay result

According to primary standard content limit and 10 batch sample measured values, this product Pericarpium Citri Reticulatae assay limit is: this product every bag contains Pericarpium Citri Reticulatae is with Hesperidin (C₂₈H₃₄O₁₅) meter, 0.62mg must not be less than.

2, Fructus Crataegi assay

2.1, instrument and reagent

Chromatograph of liquid: Waters e2695 chromatograph of liquid；ERPIXING KELI: Guizhou Runsheng Pharmaceutical Co., Ltd.；Chinese holly Rafter acid reference substance (100396-200301) is provided by Nat'l Pharmaceutical & Biological Products Control Institute；Reagent: ammonium dihydrogen phosphate, phosphoric acid are AG, water are super purified water.

2.2, assay method

2.2.1, chromatography condition

(1) chromatographic column: the pillar selecting octadecylsilane chemically bonded silica to be filler is investigated.Result shows to use Octadecylsilane chemically bonded silica is that the pillar separating effect of filler is preferable.

(2) flowing phase: compare difference flowing mutually, uses 5% ammonium dihydrogen phosphate (with phosphoric acid slow-readjustment joint pH extremely 3.0) preferably separate for flowing phase, composition citric acid to be measured and other impurity are available.

(3) detection wavelength: according to the uv absorption wavelength of citric acid reference substance, detection wavelength is set to 210nm.

(4) theoretical cam curve: according to different pillar testing results, tentative theoretical cam curve is pressed citric acid and is calculated, should not be low In 3000.

2.2.2, the preparation of need testing solution

Getting it filled composition granule, finely ground, precision weighs about 2.0g, puts in 25ml measuring bottle, and the accurate water that adds is appropriate, supersound process (power 250W, frequency 35kHz) 20 minutes, lets cool, is diluted with water to scale, shake up, and filters, and takes subsequent filtrate, crosses 0.45 μm micro- Hole filter membrane, to obtain final product.

2.2.3, the preparation of reference substance solution

Taking citric acid reference substance appropriate, accurately weighed, add water the solution making every 1ml containing 0.5mg, to obtain final product.

2.3, method validation

2.3.1, linear investigation precision weighs citric acid reference substance 0.2010g to 10ml measuring bottle and obtains A liquid (2.01mg/ml)； Take 1mlA liquid to 2ml measuring bottle and obtain B liquid (1.005mg/ml)；Take 2mlA liquid to 5ml measuring bottle and obtain C liquid (0.804mg/ml)；Take 1.5mlA liquid to 5ml measuring bottle obtains D liquid (0.603mg/ml)；Take 1ml B liquid to 2ml measuring bottle and obtain E liquid (0.402mg/ml)；Take 1ml E liquid to 2ml measuring bottle obtains F liquid (0.201mg/ml)；Take 1ml F liquid to 2ml measuring bottle and obtain G liquid (0.1005mg/ml), B, C, D, E, F, G divides and takes 20 μ l sample introductions.Record chromatograph, with peak area as vertical coordinate, is abscissa mapping with sample size (μ g), calculates regression equation For A=146399.4943C-65747.9151 (r=0.9998), regression equation matching is crossed the linear equation of initial point and is: A= 141811.156C；Being analyzed by one need testing solution sample introduction, gained peak area substitutes into two formulas respectively and calculates, and relative deviation is 3.27%, therefore it is believed that standard curve crosses initial point, regression equation no intercept.Thus assay can use one point external standard method meter Calculate.Citric acid linear relationship between 2.02 μ g～20.1 μ g is good.Concrete outcome is shown in Table 11 and Fig. 7.

Table 11 citric acid linear relationship investigates (n=2)

2.3.2, precision test

Take same citric acid reference substance solution, repeat sample introduction 6 times, record chromatograph, the results are shown in Table 12 and precision collection of illustrative plates, RSD is 0.19%, and good precision is described.

Table 12 precision test

2.3.3 replica test

Take a sample as described in text, prepare 6 parts of need testing solutions, respectively sample introduction, record chromatograph, calculate, the results are shown in Table 13, RSD is 1.01%, and good repeatability is described.

Table 13 replica test

2.3.4 recovery test

Using sample-adding to reclaim, accurate absorption has measured the sample 1g (4.007mg/g) of content, adds citric acid reference substance In right amount, as described in text, chromatographic condition measures, and calculates the response rate, the results are shown in Table 14, and response rate collection of illustrative plates, the response rate of citric acid is 102.93%, RSD are 0.92%, illustrate that this law has the good response rate.

Table 14 recovery test

2.3.5, specificity

Take ERPIXING KELI and negative sample and prepare sample solution and negative sample solution by need testing solution preparation method, By above-mentioned chromatographic condition, take reference substance solution, sample solution, negative sample solution injection chromatograph of liquid, sample solution respectively Chromatograph is equipped with corresponding chromatographic peak in reference substance solution chromatograph corresponding positions, and negative noiseless, and result is shown in Figure 11, Figure 12 and Tu 13。

2.3.6, ruggedness,

2.3.6.1, the selection of sample extraction solvent

Get it filled composition granule, finely ground, take 4 parts, every part of about 2g, accurately weighed, put in 25ml measuring bottle, be soluble according to organic acid Water, methanol, the character of ethanol, therefore precision addition suitable quantity of water, 1% sodium bicarbonate water, methanol, ethanol respectively, weighed weight, ultrasonic Process 1 hour, let cool, respectively with water, 1% sodium bicarbonate water, methanol, ethanol dilution to scale, shake up, filter, take subsequent filtrate, Cross 0.45 μm microporous filter membrane, as need testing solution.Take respectively above-mentioned solution each 20 μ l inject chromatograph of liquid, by methanol, The poor content that cannot calculate of chromatogram separating effect that ethanol extraction obtains, therefore water and 1% sodium bicarbonate water are compared, meter Calculating, the results are shown in Table 15, this product is adopted and is used water as Extraction solvent, and content extracts more complete.

The selection of table 15 Extraction solvent

2.3.6.2, the selection of sample extraction method

Get it filled composition granule, finely ground, take 3 parts, every part of about 2g, accurately weighed, precision adds 25ml water respectively, passes through back respectively Stream, ultrasonic, soak extraction, weighed weight, refluxes 1 hour, ultrasonic 1 hour, soaks 1 hour, let cool, supply less loss with water respectively Weight, filters, and takes subsequent filtrate, crosses 0.45 μm microporous filter membrane, as need testing solution.Take each 20 μ l of above-mentioned solution respectively and inject liquid Chromatography, calculates, the results are shown in Table 16, backflow, ultrasonic, soak the content that three kinds of extracting method are surveyed and be more or less the same, therefore select Simplicity, the preferable supersound extraction of effect are as extracting method.

The selection of table 16 extracting method

2.3.6.3, sample extraction time effects

Get it filled composition granule, finely ground, take 4 parts, every part of about 2.0g, accurately weighed, put in 25ml measuring bottle respectively, accurate add water In right amount, ultrasonic 10,20,40,60 minutes respectively, let cool, be diluted with water to scale, shake up, filter, take subsequent filtrate, cross 0.45 μm Microporous filter membrane, as need testing solution.Take each 20 μ l of above-mentioned solution respectively and inject chromatograph of liquid, calculate, the results are shown in Table 17, 10,20,40,60 minutes measured content is more or less the same, and in order to ensure to extract completely, therefore the selective extraction time is 20 minutes.

Table 17 extraction time affects

Extracting method is defined as: composition granule of getting it filled, finely ground, and precision weighs about 2.0g, puts in 25ml measuring bottle, accurate addition water In right amount, supersound process (power 250W, frequency 30kHz) 20 minutes, let cool, be diluted with water to scale, shake up, filter, take continuous filter Liquid, crosses 0.45 μm microporous filter membrane, to obtain final product.

2.3.6.4, the impact of different brands pillar

Using different brands octadecylsilane chemically bonded silica is the pillar of filler, determines respectively in ERPIXING KELI The content of citric acid, investigates the impact of the chromatographic column of different brands.Assay the results are shown in Table 18.Result shows different brands ten Eight alkyl silane bonded silica gels be filler chromatographic column on assay without impact.

The measurement result of table 18 different brands chromatographic column

2.3.6.5, sample stability test

Take ERPIXING KELI, prepare by need testing solution preparation method, measure citric acid peak area by above-mentioned chromatographic condition, The results are shown in Table 19.

Table 19 sample stability is tested

2.4.10 batch content sample determination:

The results are shown in Table 20.

Table 20 10 batch sample assay result

According to 10 batch sample measured values, this product Fructus Crataegi assay limit is: this product every bag contains Fructus Crataegi with citric acid (C₆H₈O₇) meter, 6.0mg must not be less than.

The beneficial effects of the present invention is: the invention provides the detection method of a kind of ERPIXING KELI, including with effectively Composition Hesperidin and citric acid are index, use high effective liquid chromatography for measuring Hesperidin and the content of citric acid；And medicine In thin-layer identification method to Fructus Crataegi, Pericarpium Citri Reticulatae, Rhizoma Dioscoreae and Endothelium Corneum Gigeriae Galli；Described detection method is accurate, highly sensitive, reproducible, Testing result is stable, can effectively control the quality of ERPIXING KELI, both be more beneficial for manufacturer and supervisory and management department to product The monitoring of quality, it is also possible to the treatment for medical department and patient provides preferably guarantee.

Accompanying drawing explanation

When Fig. 1 is hawthorn thin-layer discriminating, during specificity test, under daylight, inspect figure；1-20151001,2-20151002, 3-20151003,4-negative sample, 5-Fructus Crataegi control medicinal material, 6-ursolic acid reference substance；

When Fig. 2 is hawthorn thin-layer discriminating, during specificity test, under ultra-violet lamp (365nm), inspect figure；1-20151001, 2-20151002,3-20151003,4-negative sample, 5-Fructus Crataegi control medicinal material, 6-ursolic acid reference substance；

When Fig. 3 is hawthorn thin-layer discriminating, during specificity test, under daylight, inspect figure；1-20151001,2-20151002, 3-20151003,4-negative sample, 5-Pericarpium Citri Reticulatae control medicinal material, 6-Hesperidin reference substance；

When Fig. 4 is the discriminating of Rhizoma Dioscoreae thin layer；During specificity test, under daylight, inspect result figure, 1-20151001,2- 20151002,3-20151003,4-Rhizoma Dioscoreae control medicinal material, 5-negative sample；

When Fig. 5 is the discriminating of Endothelium Corneum Gigeriae Galli thin layer, during specificity test, under daylight, inspect result figure；1-20151001,2- 20151002,3-20151003,4-Endothelium Corneum Gigeriae Galli control medicinal material, 5-negative sample；

Fig. 6 is Hesperidin canonical plotting, X-axis be sample size (μ g), Y-axis be peak area；

Fig. 7 is citric acid canonical plotting, X-axis be sample size (μ g), Y-axis be peak area；

When Fig. 8 is content of hesperidin mensuration, Hesperidin reference substance specificity lab diagram；

When Fig. 9 is content of hesperidin mensuration, Hesperidin test sample specificity lab diagram；

When Figure 10 content of hesperidin measures, Hesperidin negative sample specificity lab diagram；

When Figure 11 is citric acid assay, citric acid reference substance specificity lab diagram；

When Figure 12 is citric acid assay, citric acid test sample specificity lab diagram；

When Figure 13 is citric acid assay, citric acid negative sample specificity lab diagram.

Below in conjunction with embodiment, the present invention is further illustrated

Detailed description of the invention

Embodiment: the detection method of granule

Granular formulations: Fructus Crataegi 320g, Fructus Hordei Germinatus 320g, Endothelium Corneum Gigeriae Galli 240g, Rhizoma Dioscoreae 240g, Semen Coicis 160g, Semen Lablab Album 240g, Pericarpium Citri Reticulatae 96g and Poria 96g.

Technique: above eight tastes, Fructus Crataegi, tangerine peel powder are broken into coarse powder, use 65% ethanol as solvent, after impregnating 24 hours, carry out Percolation, collects liquid of filtering, and diacolation liquid recycling ethanol, when being concentrated into 55-65 DEG C, relative density is the thick paste of 1.35-1.40, standby；Remaining The Six-element medical material boilings such as Fructus Hordei Germinatus three times, each 1 hour, collecting decoction, filter, it is 1.10 that filtrate is concentrated into relative density The clear paste of (60 DEG C), adds equivalent ethanol and makes precipitation, takes supernatant, reclaims ethanol, and being concentrated into relative density is the thick of 1.35-1.40 Cream；With above-mentioned Fructus Crataegi, the mixing of Pericarpium Citri Reticulatae thick paste, add sucrose in right amount, make granule, be dried, to obtain final product.

Usage and consumption: warm boiled water.1～5 years old one time 1 bag, 3 times on the one；5 years old with last 2 bags, 3 times on the one；10 It is a course for the treatment of.

Specification: every packed 2.5g.

Detection method:

Character: medicine is the granule of brown color；Feeble QI is fragrant, sweet and sour, micro-hardship.

Differentiate:

(1) get it filled composition granule content, finely ground after, take 8g and add 25ml ether, be heated to reflux 1 hour, filter, filtrate is waved Dry, residue adds methanol 1m1 makes dissolving, as need testing solution；Separately take Fructus Crataegi control medicinal material 0.2g, finely ground after, take 8g and add 25ml Ether, is heated to reflux 1 hour, filters, and filtrate volatilizes, and residue adds methanol 1m1 makes dissolving, prepares control medicinal material solution；Take Bears again Fruit acid reference substance is appropriate, adds methanol and makes every 1ml solution containing 0.1mg, as reference substance solution；According to Chinese Pharmacopoeia thin layer color Spectrometry is tested, and draws above-mentioned three all solution each 5～10 μ l, puts respectively on same silica gel g thin-layer plate, with toluene: acetic acid second Ester: formic acid=20: be developing solvent at 4: 0.5, launches, takes out, dry, and spray is with ethanol solution of sulfuric acid (3 → 10), 105 DEG C of heating Clear to spot development, put respectively and inspect under daylight or 365nm ultra-violet lamp, in test sample chromatograph, with control medicinal material chromatograph On corresponding position, the principal spot of aobvious same color or fluorescence principal spot；On position corresponding with reference substance chromatograph, aobvious identical The speckle of color or fluorescence speckle.

(2) get it filled composition granule content, finely ground after, take 10g, add methanol 30ml, be heated to reflux 1 hour, filter, filtrate is waved Dry, the residue 10ml that adds water makes dissolving, is transferred in separatory funnel, extracts 2 times with ethyl acetate shaking, each each 10ml, merge Acetic acid ethyl acetate extract, water-bath is evaporated, and residue adds methanol 2ml makes dissolving, as need testing solution；Separately take Pericarpium Citri Reticulatae control medicinal material 1g, is made in the same way of control medicinal material solution；Take Hesperidin reference substance more appropriate, add methanol and make saturated solution, molten as reference substance Liquid；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned three kinds of solution each 2～5 μ l, put respectively and use 0.5% hydrogen in same On silica gel g thin-layer plate prepared by sodium hydroxide solution, with ethyl acetate: methanol: water=100: 17: 13 as developing solvent, Zhan Zhiyue 3cm, takes out, dries, then with toluene: ethyl acetate: formic acid: water=20: the upper solution of 10: 1: 1, as developing solvent, is opened up to 8cm, Taking out, dry, spray is with 5% aluminum chloride ethanol solution, and being placed in wavelength is to inspect under 365nm ultra-violet lamp；In test sample chromatograph, On position corresponding with control medicinal material chromatograph, the fluorescence principal spot of aobvious same color；In position corresponding with reference substance chromatograph On, the fluorescence speckle of aobvious same color.

(3) get it filled composition granule content, finely ground, take 5g, add water 30ml, steeped overnight, and aqueous solution moves together with residue Put in separatory funnel, add the shaking of water saturated n-butyl alcohol and extract 3 times, each 20ml, divide and take n-butyl alcohol liquid, be evaporated, residue adds suitable Amount methanol makes dissolving, is added on the neutral alumina column of 100-120 mesh, 5g, internal diameter 10-15mm, washes with 10% methanol 100ml De-, to collect eluent, be evaporated, residue adds 1ml70% ethanol makes dissolving, as need testing solution；Separately take Endothelium Corneum Gigeriae Galli control medicinal material 1g, add water 30ml, decocts 2 hours, and aqueous solution, together with in residue dislocation separatory funnel, is made in the same way of control medicinal material solution； Test according to Chinese Pharmacopoeia thin layer chromatography, draw above two solution each 3～5 μ l, put respectively in same silica gel g thin-layer plate On, with n-butyl alcohol: glacial acetic acid: water=9: 3: 1 as developing solvent, the lamellae after pre-equilibration point sample 15 minutes, launches, takes out, dry in the air Dry, spray, with ninhydrin solution, is heated to spot development at 110 DEG C clear；In test sample chromatograph, corresponding to control medicinal material chromatograph Position on, the principal spot of aobvious same color.

Every regulation relevant under (four annex I C of " Chinese Pharmacopoeia " version in 2015) granule item should be met.

Assay:

(1) Hesperidin:

This product every bag contains Pericarpium Citri Reticulatae with Hesperidin (C₂₈H₃₄O₁₅) meter, 0.62mg must not be less than.

(2) citric acid: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring:

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；With 5% biphosphate Ammonium salt solution (saving pH to 3.0 with phosphoric acid slow-readjustment) is flowing phase, and column temperature is 25 DEG C；Detection wavelength is 210nm；Number of theoretical plate presses citron Acid peak calculates and should be not less than 3000；

This product every bag contains Fructus Crataegi with citric acid (C₆H₈O₇) meter, 6.0mg must not be less than.

Claims

1. a detection method for ERPIXING KELI, described granule is mainly by the Fructus Crataegi 300-340 part calculated by weight, Fructus Hordei Germinatus 300-340 part, Endothelium Corneum Gigeriae Galli 220-260 part, Rhizoma Dioscoreae 220-260 part, Semen Coicis 140-180 part, Semen Lablab Album 220-260 part, Pericarpium Citri Reticulatae 86-106 part and Poria 86-106 part are made as follows: above eight tastes, and Fructus Crataegi, tangerine peel powder are broken into coarse powder, use 65% ethanol Making solvent, after impregnating 24 hours, carry out percolation, collect liquid of filtering, diacolation liquid recycling ethanol, when being concentrated into 55-65 DEG C, relative density is 1.35-1.40 thick paste, standby；The Six-element medical material boilings such as remaining Fructus Hordei Germinatus three times, each 1 hour, collecting decoction, filter, When filtrate is concentrated into 55-65 DEG C, relative density is the clear paste of 1.10, adds equivalent ethanol and makes precipitation, takes supernatant, reclaims ethanol, dense It is reduced to the thick paste that relative density is 1.35-1.40；With above-mentioned Fructus Crataegi, the mixing of Pericarpium Citri Reticulatae thick paste, add sucrose in right amount, make granule, It is dried, to obtain final product；It is characterized in that: described detection method includes discrimination method and content assaying method；Described discrimination method includes The thin layer of Fructus Crataegi, Pericarpium Citri Reticulatae, Rhizoma Dioscoreae or Endothelium Corneum Gigeriae Galli is differentiated detection；Described content assaying method includes Hesperidin or citric acid Assay.

2. the detection method of ERPIXING KELI as claimed in claim 1, it is characterised in that: the discrimination method of described Fructus Crataegi is: Get it filled composition granule, finely ground after, take 4-12g and add 20-30ml ether, be heated to reflux 0.5-1.5 hour, filter, filtrate volatilizes, residue Add methanol 0.5-1.5m1 and make dissolving, as need testing solution；Separately take Fructus Crataegi control medicinal material 0.1-0.3g, finely ground after, take 4-12g Adding 20-30ml ether, be heated to reflux 0.5-1.5 hour, filter, filtrate volatilizes, and residue adds methanol 0.5-1.5m1 makes dissolving, system Obtain control medicinal material solution；Take ursolic acid reference substance more appropriate, add methanol and make every 0.5-1.5ml solution containing 0.05-0.15mg, As reference substance solution；According to Chinese Pharmacopoeia thin layer chromatography test, draw above-mentioned three all solution each 5-10 μ l, put respectively in On same silica gel g thin-layer plate, with toluene: ethyl acetate: formic acid=15-25: 2-6: 0.1-1.0, as developing solvent, launches, and takes out, Drying, spray with ethanol solution of sulfuric acid, in ethanol solution of sulfuric acid, sulphuric acid is 2-4:9-11 with the volume ratio of ethanol；105 DEG C of heating Clear to spot development, put respectively and inspect under daylight or 360-370nm ultra-violet lamp, in test sample chromatograph, with control medicinal material On the corresponding position of chromatograph, the principal spot of aobvious same color or fluorescence principal spot；On position corresponding with reference substance chromatograph, aobvious The speckle of same color or fluorescence speckle.

3. the detection method of ERPIXING KELI as claimed in claim 2, it is characterised in that: the discrimination method of described Fructus Crataegi is: Get it filled composition granule, finely ground after, take 8g and add 25ml ether, be heated to reflux 1 hour, filter, filtrate volatilizes, and residue adds methanol 1m1 to be made Dissolve, as need testing solution；Separately take Fructus Crataegi control medicinal material 0.2g, finely ground after, take 8g and add 25ml ether, be heated to reflux 1 hour, Filtering, filtrate volatilizes, and residue adds methanol 1m1 makes dissolving, prepares control medicinal material solution；Take ursolic acid reference substance more appropriate, add first Every 1ml solution containing 0.1mg made by alcohol, as reference substance solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned three All solution each 5～10 μ l, puts on same silica gel g thin-layer plate, respectively with toluene: ethyl acetate: formic acid=20: be at 4: 0.5 Developing solvent, launches, and takes out, dries, and sprays with ethanol solution of sulfuric acid, and in ethanol solution of sulfuric acid, sulphuric acid is 3 with the volume ratio of ethanol: 10；It is heated to spot development at 105 DEG C clear, puts respectively and inspect under daylight or 365nm ultra-violet lamp, in test sample chromatograph, On position corresponding with control medicinal material chromatograph, the principal spot of aobvious same color or fluorescence principal spot；Corresponding to reference substance chromatograph Position on, the speckle of aobvious same color or fluorescence speckle.

4. the detection method of ERPIXING KELI as claimed in claim 1, it is characterised in that: the discrimination method of described Pericarpium Citri Reticulatae is: Get it filled composition granule, finely ground after, take 5-15g, add methanol 25-35ml, be heated to reflux 0.5-1.5 hour, filter, filtrate volatilizes, residual The slag 5-15ml that adds water makes dissolving, is transferred in separatory funnel, extracts 2-3 time with ethyl acetate shaking, each each 5-15ml, merging Acetic acid ethyl acetate extract, water-bath is evaporated, and residue adds methanol 1-4ml makes dissolving, as need testing solution；Separately take Pericarpium Citri Reticulatae comparison medicine Material 0.5-1.5g, is made in the same way of control medicinal material solution；Take Hesperidin reference substance more appropriate, add methanol and make saturated solution, as Reference substance solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned three kinds of solution each 2-5 μ l, put respectively in same use On silica gel g thin-layer plate prepared by 0.1-1.0% sodium hydroxide solution, with ethyl acetate: methanol: water=95-105: 15-19: 10- 16 is developing solvent, opens up to 1-6cm, takes out, dry, then with toluene: ethyl acetate: formic acid: water=15-25: 5-15: 0.5-1.5: The upper solution of 0.5-1.5 is developing solvent, opens up to 5-10cm, takes out, dry, and spray, with 1-10% aluminum chloride ethanol solution, is put Inspect under wavelength is 360-370nm ultra-violet lamp；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious The fluorescence principal spot of same color；On position corresponding with reference substance chromatograph, the fluorescence speckle of aobvious same color.

5. the detection method of ERPIXING KELI as claimed in claim 1, it is characterised in that: the discrimination method of described Endothelium Corneum Gigeriae Galli For: composition granule of getting it filled, finely ground, take 1-10g, add water 25-35ml, steeped overnight, and aqueous solution leaks together with residue dislocation separatory In bucket, add the shaking of water saturated n-butyl alcohol and extract 2-4 time, each 15-25ml, divide and take n-butyl alcohol liquid, be evaporated, residue adds appropriate first Alcohol makes dissolving, is added on the neutral alumina column of 100-120 mesh, 1-10g, internal diameter 10-15mm, with 5-15% methanol 90-110ml Eluting, collects eluent, is evaporated, and residue adds 0.5-1.5ml, 65-75% ethanol makes dissolving, as need testing solution；Separately take chicken Endothelium corneum control medicinal material 0.5-1.5g, add water 25-35ml, decocts 1-3 hour, and aqueous solution is together with residue dislocation separatory funnel In, it is made in the same way of control medicinal material solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above two solution each 3～5 μ l, Put respectively on same silica gel g thin-layer plate, with n-butyl alcohol: glacial acetic acid: water=5-15: 1-6: 0.5-1.5 as developing solvent, pre-equilibration Lamellae after point sample 10-20 minute, launches, and takes out, dries, and spray, with ninhydrin solution, is heated to spot development at 110 DEG C clear Clear；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the principal spot of aobvious same color.

6. the detection method of ERPIXING KELI as claimed in claim 5, it is characterised in that: the discrimination method of described Endothelium Corneum Gigeriae Galli For: composition granule of getting it filled, finely ground, take 5g, add water 30ml, steeped overnight, and aqueous solution, together with in residue dislocation separatory funnel, adds The shaking of water saturated n-butyl alcohol is extracted 3 times, and each 20ml divides and takes n-butyl alcohol liquid, is evaporated, and residue adds proper amount of methanol makes dissolving, adds In 100-120 mesh, 5g, internal diameter 10-15mm neutral alumina column on, with 10% methanol 100ml eluting, collect eluent, steam Dry, residue adds 1ml70% ethanol makes dissolving, as need testing solution；Separately taking Endothelium Corneum Gigeriae Galli control medicinal material 1g, add water 30ml, decocts 2 Hour, aqueous solution, together with in residue dislocation separatory funnel, is made in the same way of control medicinal material solution；According to Chinese Pharmacopoeia thin layer color Spectrometry is tested, and draws above two solution each 3～5 μ l, puts respectively on same silica gel g thin-layer plate, with n-butyl alcohol: glacial acetic acid: Water=9: be developing solvent at 3: 1, the lamellae after pre-equilibration point sample 15 minutes launches, takes out, dry, sprays with ninhydrin solution, 110 DEG C to be heated to spot development clear；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious same color Principal spot.

7. the detection method of ERPIXING KELI as claimed in claim 1, it is characterised in that: described content of hesperidin assay method For: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring:

Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；With methanol: glacial acetic acid: water =30-40: 1-8: 55-65 is flowing phase；Column temperature is 33-37 DEG C；Detection wavelength is 280-286nm；Number of theoretical plate presses Hesperidin Peak calculates should be not less than 3000；

The preparation of need testing solution: composition granule content of getting it filled, mixing, finely ground, take 0.2-0.6g, accurately weighed, put tool plug cone In shape bottle, accurate addition methanol 20-30ml, close plug, weighed weight, supersound process, sonification power is 200-300W, ultrasonic Process frequency is 30-40kHz, and sonication treatment time is 25-35 minute, lets cool, more weighed weight, supplies the weight of less loss with methanol Amount, shakes up, and filters, takes subsequent filtrate, to obtain final product；

Algoscopy: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, to obtain final product.

8. the detection method of ERPIXING KELI as claimed in claim 7, it is characterised in that: described content of hesperidin assay method For: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring:

Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；With methanol: glacial acetic acid: water Be flowing phase at=35: 4: 61；Column temperature is 35 DEG C；Detection wavelength is 283nm；Number of theoretical plate is calculated should be not less than by Hesperidin peak 3000；

The preparation of reference substance solution: take Hesperidin reference substance appropriate, accurately weighed, add methanol and make molten containing 0.01mg of every 1ml Liquid, to obtain final product；

The preparation of need testing solution: composition granule content of getting it filled, mixing, finely ground, take 0.4g, accurately weighed, put tool plug conical flask In, accurate addition methanol 25ml, close plug, weighed weight, supersound process, sonification power is 250W, and supersound process frequency is 35kHz, sonication treatment time is 30 minutes, lets cool, more weighed weight, supplies the weight of less loss with methanol, shakes up, and filters, takes Subsequent filtrate, to obtain final product；

9. the detection method of ERPIXING KELI as claimed in claim 1, it is characterised in that: described citric acid content assaying method For: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring:

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；Number of theoretical plate presses citric acid Peak calculates should be not less than 3000；

The preparation of reference substance solution: take citric acid reference substance appropriate, accurately weighed, add water and make every 0.5-1.5ml containing 0.1- The solution of 0.6mg, to obtain final product；

The preparation of need testing solution: composition granule content of getting it filled, finely ground, precision weighs 1-4g, puts in 20-30ml measuring bottle, accurate Adding water appropriate, supersound process, sonification power is 200-300W, and supersound process frequency is 30-40kHz, during supersound process Between be 15-25 minute, let cool, be diluted with water to scale and shake up, filter, take subsequent filtrate, cross 0.35-0.55 μm microporous filter membrane, i.e. ?.

Algoscopy: precision draws reference substance solution and need testing solution each 15-25 μ l respectively, injects chromatograph of liquid, measures, i.e. ?.

10. the detection method of ERPIXING KELI as claimed in claim 1, it is characterised in that:

The discrimination method of described Fructus Crataegi is: after composition granule of getting it filled is finely ground, takes 8g and adds 25ml ether, is heated to reflux 1 hour, filters, Filtrate volatilizes, and residue adds methanol 1m1 makes dissolving, as need testing solution；Separately take Fructus Crataegi control medicinal material 0.2g, finely ground after, take 8g Adding 25ml ether, be heated to reflux 1 hour, filter, filtrate volatilizes, and residue adds methanol 1m1 makes dissolving, prepares control medicinal material solution； Take ursolic acid reference substance more appropriate, add methanol and make every 1ml solution containing 0.1mg, as reference substance solution；According to Chinese Pharmacopoeia Thin layer chromatography is tested, and draws above-mentioned three all solution each 5～10 μ l, puts respectively on same silica gel g thin-layer plate, with toluene: Ethyl acetate: formic acid=20: be developing solvent at 4: 0.5, launches, takes out, dry, and spray is with ethanol solution of sulfuric acid, ethanol solution of sulfuric acid Middle sulphuric acid is 3:10 with the volume ratio of ethanol；It is heated to spot development at 105 DEG C clear, puts daylight or 365nm ultraviolet light respectively Inspect under lamp, in test sample chromatograph, on position corresponding with control medicinal material chromatograph, the principal spot of aobvious same color or fluorescence master Speckle；On position corresponding with reference substance chromatograph, the speckle of aobvious same color or fluorescence speckle；

The discrimination method of described Pericarpium Citri Reticulatae is: composition granule content of getting it filled, finely ground after, take 10g, add methanol 30ml, be heated to reflux 1 little Time, filtering, filtrate volatilizes, and the residue 10ml that adds water makes dissolving, is transferred in separatory funnel, extracts 2 times with ethyl acetate shaking, often Secondary each 10ml, combined ethyl acetate extracting solution, water-bath is evaporated, residue adds methanol 2ml makes dissolving, as need testing solution；Separately Take Pericarpium Citri Reticulatae control medicinal material 1g, be made in the same way of control medicinal material solution；Take Hesperidin reference substance more appropriate, add methanol and make saturated molten Liquid, as reference substance solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned three kinds of solution each 2～5 μ l, point respectively On the same silica gel g thin-layer plate prepared with 0.5% sodium hydroxide solution, with ethyl acetate: methanol: water=100: be at 17: 13 Developing solvent, opens up to about 3cm, takes out, dry, then with toluene: ethyl acetate: formic acid: water=20: the upper solution of 10: 1: 1 is for exhibition Opening agent, open up to 8cm, take out, dry, spray is with 5% aluminum chloride ethanol solution, and being placed in wavelength is to inspect under 365nm ultra-violet lamp； In test sample chromatograph, on position corresponding with control medicinal material chromatograph, the fluorescence principal spot of aobvious same color；With reference substance color Compose on corresponding position, the fluorescence speckle of aobvious same color；

The discrimination method of described Endothelium Corneum Gigeriae Galli is: composition granule of getting it filled, finely ground, takes 5g, and add water 30ml, steeped overnight, aqueous solution together with In residue dislocation separatory funnel together, add the shaking of water saturated n-butyl alcohol and extract 3 times, each 20ml, divides and takes n-butyl alcohol liquid, steam Dry, residue adds proper amount of methanol makes dissolving, is added on the neutral alumina column of 100-120 mesh, 5g, internal diameter 10-15mm, uses 10% first Alcohol 100ml eluting, collects eluent, is evaporated, and residue adds 1ml70% ethanol makes dissolving, as need testing solution；Separately take Endothelium Corneum Gigeriae Galli Control medicinal material 1g, add water 30ml, decocts 2 hours, and aqueous solution, together with in residue dislocation separatory funnel, is made in the same way of comparison medicine Material solution；Test according to Chinese Pharmacopoeia thin layer chromatography, draw above two solution each 3～5 μ l, put respectively in same silica gel G On lamellae, with n-butyl alcohol: glacial acetic acid: water=9: 3: 1 as developing solvent, the lamellae after pre-equilibration point sample 15 minutes, launches, takes Going out, dry, spray, with ninhydrin solution, is heated to spot development at 110 DEG C clear；In test sample chromatograph, with control medicinal material color Compose on corresponding position, the principal spot of aobvious same color；

Algoscopy: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, to obtain final product； Described citric acid content assaying method is: according to Chinese Pharmacopoeia high effective liquid chromatography for measuring:

Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica；Molten with 5% ammonium dihydrogen phosphate Liquid, is flowing phase with phosphoric acid slow-readjustment joint pH to 3.0, and column temperature is 25 DEG C；Detection wavelength is 210nm；Number of theoretical plate presses citric acid peak Calculating should be not less than 3000；

The preparation of reference substance solution: take citric acid reference substance appropriate, accurately weighed, add water the solution making every 1ml containing 0.3mg, Obtain；

The preparation of need testing solution: composition granule content of getting it filled, finely ground, precision weighs 2.0g, puts in 25ml measuring bottle, accurate addition Water is appropriate, supersound process, and sonification power is 250W, and supersound process frequency is 35kHz, and sonication treatment time is 20 minutes, Let cool, be diluted with water to scale and shake up, filter, take subsequent filtrate, cross 0.45 μm microporous filter membrane, to obtain final product.

Algoscopy: precision draws reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid, measures, to obtain final product.