CN106153812A - A kind of detection method of the Chinese medicine preparation treating endometriosis - Google Patents

Abstract

The invention discloses the detection method of a kind of Chinese medicine preparation treating endometriosis, including differentiating and assay project；Discriminating is to Radix Paeoniae Rubra, Cortex Moutan, Ramulus Cinnamomi, the Radix Aucklandiae, Fructus Corni, Rhizoma Curcumae, the indentification by TLC of Poria；Assay is with Radix Paeoniae Rubra, the content of Herba Polygoni cymosi in Preparations by HPLC.The Chinese medicine preparation detection method of the treatment endometriosis of the present invention, specificity is strong, and precision is high, favorable reproducibility, and the response rate is high, and measurement result is accurate, can effectively control drug quality, so that it is guaranteed that the stable and clinical application of product quality is safely, effectively.

Description

A kind of detection method of the Chinese medicine preparation treating endometriosis

Technical field

The present invention relates to the detection method of a kind of Chinese medicine preparation treating endometriosis, belong to traditional Chinese medicine technology neck Territory.

Background technology

Endometriosis refers to the endometrium with growth function, is covered in other portions of the health beyond cavity of uterus , there is a kind of gynaecopathia of cyclic hemorrhage repeatedly simultaneously in position.Ectopic Endometrium functionally can be along with the change of estrogen level Change, also can produce significantly change, not only have the body of gland of inner membrance, also have inner membrance interstitial around.Endometriosis is main Occurring at pelvic organ, cervix uteri, vagina, perineum and abdominal incision are likely to there will be, and have the strongest transformation and business growth ability, for Common gynecological disease disease.Gynecopathy is common in the women of child-bearing age more, and its sickness rate ascendant trend in recent years is very fast, the wherein women of child-bearing age Account for more than 10%, and relapse rate is high.

Chinese medicine is to be its fundamental characteristic with determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs to the diagnoses and treatment of " gynecopathy ", and Chinese medicine is thought, " interior different Disease " main symptom be syndrome of blood stasis, and through the whole process of disease.The disease initial stage, sick wound for a long time just, caused void because of real based on excess syndrome, Rare pure deficiency syndrome, master shows as simulataneous insufficiency and excessive disease.True person, premenstrual dysmenorrhea, menstrual period sharp ache, tenderness.Simulataneous insufficiency and excessive person, warp Phase dysmenorrhea, through after pain gesture the most slow.Because trembling with fear and stasis of blood person, for cold type of pain, angor, pain alleviated while getting warmth；Because of thermic stasis of blood person, for causalgia, obtain burning pain Increase；Because of stagnation of QI person, swollen pendant is had a pain；Blood stasis notably, for twinge.Because the stagnation of QI or cold coagulation cause stasis of blood person, hypomenorrhea, color is purple dark, has clot. Because of hot or damp and hot and stasis of blood person, many through amount, color depth is red, and matter is thick clot.Blood stasis due to qi deficiency person, also many also few through amount, color is light dim, and matter is dilute Folder clot.By dialectical to this disease of the traditional Chinese medical science, it is generally divided into syndrome of qi stagnation and blood stasis, cold blood stasis card, blood stasis due to renal deficiency card, damp and hot blood stasis The types such as card.

Chinese medicine has its original advantage on treatment endometriosis, and Chinese medicine is to endometriosis not only It is only to play its therapeutical effect, also can preferably regulate the many aspects such as endocrine and immunity of body simultaneously.Select Chinese medicine, Take modern preferably extraction process and preparation technology, prepare medicine, provide one really by the treatment for endometriosis Medicable Chinese medicine new varieties, provide new medicine for this patient.Therefore, the Chinese medicine to treatment endometriosis Preparation detects, and controls drug quality, ensures drug safety, it appears particularly necessary.

Summary of the invention

For solving the deficiencies in the prior art, it is an object of the invention to provide a kind of Chinese medicine treating endometriosis The detection method of preparation, measurement result is accurate, can effectively control drug quality.

The Chinese medicine preparation for the treatment of endometriosis of the present invention is: count by weight, by Sanguis Draxonis 53～198 Part, Radix Et Rhizoma Rhei 99～495 parts, Fructus Corni 198～396 parts, Radix Angelicae Sinensis 198～396 parts, Cortex Moutan 198～396 parts, the Radix Aucklandiae 99～330 Part, Rhizoma Corydalis 99～330 parts, Radix Paeoniae Rubra 198～396 parts, Poria 293～495 parts, Rhizoma Curcumae 173～297 parts, Herba Polygoni cymosi 105～ 990 parts, Ramulus Cinnamomi 99～330 parts and Rhizoma Sparganii 165～330 parts are made.Its preparation method is: weighs Herba Polygoni cymosi, Fructus Corni according to quantity, prolong Rhizoma Corydalis and rhubarb medicinal material, add ethanol, reflux, extract, filter, obtain alcohol extract, standby；Weigh according to quantity Sanguis Draxonis, Radix Angelicae Sinensis, Cortex Moutan, The Radix Aucklandiae, Radix Paeoniae Rubra, Poria, Rhizoma Curcumae, Ramulus Cinnamomi and Rhizoma Sparganii, add water, and decocts and extracts, and filters, obtains Aqueous extracts, through concentrating under reduced pressure, obtain concentration Aqueous extracts, adds ethanol, stirs, and stands, discards precipitation, obtain precipitate with ethanol supernatant, standby；Alcohol extract and precipitate with ethanol supernatant Mixing, concentrating under reduced pressure, obtain concentrated solution, concentrated solution is vacuum dried, and obtains extractum extract, pulverizes and sieves, obtain medicated powder, adds auxiliary Various pharmaceutical preparation made by material.

In order to realize above-mentioned target, the present invention adopts the following technical scheme that:

The detection method of a kind of Chinese medicine preparation treating endometriosis, including differentiating and assay project；Mirror It not to Radix Paeoniae Rubra, Cortex Moutan, Ramulus Cinnamomi, the Radix Aucklandiae, Fructus Corni, Rhizoma Curcumae, the indentification by TLC of Poria；Assay is to use high-efficient liquid Phase chromatography measures Radix Paeoniae Rubra, the content of Herba Polygoni cymosi in preparation.

In the detection method of the Chinese medicine preparation of aforementioned therapies endometriosis, the discrimination method of Ramulus Cinnamomi is with cinnamic aldehyde Reference substance is comparison, with petroleum ether-ethyl acetate=17:3 thin layer chromatography as developing solvent that boiling range is 60～90 DEG C；The Radix Aucklandiae Discrimination method be with dehydrocostuslactone reference substance, costunolide reference substance for comparison, with cyclohexane-carboxylic acid ethyl ester-first The upper solution of acid=15:5:1 is the thin layer chromatography of developing solvent；The discrimination method of Fructus Corni is to be right with ursolic acid reference substance According to, with toluene-ethyl acetate-formic acid=20:4:0.5 thin layer chromatography as developing solvent；The discrimination method of Rhizoma Curcumae is Yi Jima Ketone reference substance is comparison, the thin layer color with petroleum ether-acetone and ethyl acetate=94:5:1 that boiling range is 30～60 DEG C as developing solvent Spectrometry；The discrimination method of Poria is with Poria control medicinal material for comparison, with toluene-ethyl acetate-formic acid=20:5:0.5 for exhibition Open the thin layer chromatography of agent.

Further, in the detection method of the Chinese medicine preparation of aforementioned therapies endometriosis, concrete discrimination method bag Include at least one in following project:

(1) Ramulus Cinnamomi takes this preparation or its content 3g, adds ethanol 10mL, close plug, soaks 20 minutes, shake constantly, filters, Filtrate is as need testing solution；Separately take cinnamic aldehyde reference substance, add ethanol and make every 1mL solution containing 1 μ L, molten as reference substance Liquid；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw need testing solution 10～15 μ L, reference substance Solution 2 μ L, puts on same silica gel g thin-layer plate, with petroleum ether-ethyl acetate=17:3 that boiling range is 60～90 DEG C for launching Agent, launches, and takes out, dries, and spray is with dinitrophenylhydrazine ethanol test solution；In test sample chromatograph, in position corresponding with reference substance chromatograph Put, the speckle of aobvious same color；

(2) Radix Aucklandiae takes this preparation or its content 4g, adds methanol 10mL, supersound process 30 minutes, filters, and takes filtrate and makees For need testing solution；Separately take dehydrocostuslactone reference substance, costunolide reference substance, add methanol and be respectively prepared every 1mL and contain The solution of 0.5mg, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, in absorption State three kinds of each 5 μ L of solution, put respectively on same silica gel g thin-layer plate, upper with cyclohexane-carboxylic acid ethyl ester-formic acid=15:5:1 Layer solution is developing solvent, launches, and takes out, dries, and spray, with 1% vanillin-sulfuric acid solution, is heated to spot development clear；Test sample In chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color；

(3) Fructus Corni takes this preparation or its content 3g, adds ethyl acetate 10mL, supersound process 15 minutes, filters, filter Liquid is evaporated, and residue adds dehydrated alcohol 2mL makes dissolving, as need testing solution；Separately take ursolic acid reference substance, add dehydrated alcohol and make Every 1mL solution containing 1mg, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, Draw each 5 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20:4: 0.5 is developing solvent, launches, takes out, dry, and spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear；Supply In test product chromatograph, on position corresponding with reference substance chromatograph, aobvious identical aubergine speckle；

(4) Rhizoma Curcumae takes this preparation or its content 3g, puts in tool plug centrifuge tube, adds the petroleum ether that boiling range is 30～60 DEG C 10mL, supersound process 20 minutes, filter, filtrate volatilizes, and residue adds dehydrated alcohol 1mL makes dissolving, as need testing solution；Separately take 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-reference substance, adds dehydrated alcohol and makes every 1mL solution containing 0.4mg, as reference substance solution；According to Chinese Pharmacopoeia 2010 One annex VI B thin layer chromatography test of version, draws each 10 μ L of above two solution, puts respectively on same silica gel g thin-layer plate, With petroleum ether-acetone and ethyl acetate=94:5:1 that boiling range is 30～60 DEG C as developing solvent, launching, take out, dry, spray is with 1% Vanillin-sulfuric acid solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, in position corresponding with reference substance chromatograph Put, the speckle of aobvious same color；

(5) Poria takes this preparation or its content 4g, and add diethyl ether 50mL, supersound process 10 minutes, filters, and filtrate is evaporated, Residue adds methanol 1mL makes dissolving, as need testing solution；Separately take Poria control medicinal material lg, be made in the same way of control medicinal material solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draws each 2 μ L of above two solution, puts respectively in same On silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20:5:0.5 as developing solvent, launch, take out, dry, spray with 2% fragrant Oxalaldehyde sulfuric acid solution-ethanol=4:1 mixed solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, with right According on the corresponding position of product chromatograph, show the principal spot of same color.

In the detection method of the Chinese medicine preparation of aforementioned therapies endometriosis, the discrimination method of Radix Paeoniae Rubra is with peoniflorin Reference substance is comparison, with chloroform-ethyl acetate-methyl alcohol-formic acid=40:5:10:0.2 thin layer chromatography as developing solvent；Paeonia suffruticosa The discrimination method of skin is with paeonol reference substance for comparison, with cyclohexane-ethyl acetate-glacial acetic acid=4:1:0.1 as developing solvent Thin layer chromatography.

Further, in the detection method of the Chinese medicine preparation of aforementioned therapies endometriosis, the discriminating side of Cortex Moutan Method is particularly as follows: take this preparation or its content 4g, and add diethyl ether l0mL, close plug, shakes 10 minutes, filters, and filtrate volatilizes, and residue adds Acetone 2mL makes dissolving, as need testing solution；Separately take paeonol reference substance, add acetone and make every 1mL solution conduct containing 2mg Reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw each 10 μ of above two solution L, puts respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate-glacial acetic acid=4:1:0.1 as developing solvent, launches, takes Going out, dry, spray, with 2% vanillin-sulfuric acid ethanol solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, with On the corresponding position of reference substance chromatograph, the speckle of aobvious same color.

Further, in the detection method of the Chinese medicine preparation of aforementioned therapies endometriosis, the discrimination method of Radix Paeoniae Rubra Particularly as follows: take this preparation or its content 3g, adding ethanol 10mL, shake 5 minutes, filter, filtrate is evaporated, and residue adds ethanol 2mL Make dissolving, as need testing solution；Separately take peoniflorin reference substance, add ethanol and make every 1mL solution containing 2mg, as reference substance Solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw each 4 μ L of above two solution, point respectively On same silica gel g thin-layer plate, with chloroform-ethyl acetate-methyl alcohol-formic acid=40:5:10:0.2 as developing solvent, launch, take out, Drying, spray, with 5% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, corresponding to reference substance chromatograph Position on, aobvious identical bluish violet speckle.

In the detection method of the Chinese medicine preparation of aforementioned therapies endometriosis, the content assaying method of Radix Paeoniae Rubra is with Chinese herbaceous peony Medicine glycosides reference substance is comparison, the high performance liquid chromatography being the phase that flows with methanol-0.05mol/L potassium dihydrogen phosphate=40:65 Method；The content assaying method of Herba Polygoni cymosi is with Quercetin reference substance for comparison, with methanol-0.4% phosphoric acid solution=50:50 for stream The high performance liquid chromatography of dynamic phase.

Further, in the detection method of the Chinese medicine preparation of aforementioned therapies endometriosis, concrete assay Method includes at least one in following project:

(1) Radix Paeoniae Rubra is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol- 0.05mol/L potassium dihydrogen phosphate=40:65 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 230nm, number of theoretical plate should be not less than 3000 based on peoniflorin peak；

It is appropriate that the preparation of reference substance solution takes peoniflorin reference substance, accurately weighed, adds methanol and makes every 1mL containing peoniflorin 30 The solution of μ g, to obtain final product；

The preparation of need testing solution takes this preparation or its content 1g, accurately weighed, to 50mL conical flask, adds methanol Extraction solvent 25mL, supersound extraction 10min, take out, filter, take subsequent filtrate, to obtain final product；

Algoscopy precision respectively draws reference substance solution and each 10 μ L of need testing solution, injects chromatograph of liquid, measures, i.e. ?；

(2) Herba Polygoni cymosi is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol-0.4% phosphorus Acid solution=50:50 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 360nm；Number of theoretical plate presses Quercetin Peak meter should be not less than 3000；

It is appropriate that the preparation of reference substance solution takes Quercetin reference substance, accurately weighed, adds methanol and makes every 1mL containing Quercetin 30 The solution of μ g, to obtain final product；

The preparation of need testing solution takes this preparation or its content 2g, accurately weighed, to round-bottomed flask, and addition hydrochloric acid: The mixed solution 50mL of methanol=1:4, weighed weight, put 90 DEG C of water-bath reflux, extract, 1 hour, take out, cooling, weighed weight, Supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；

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

In the detection method of the Chinese medicine preparation of aforementioned therapies endometriosis, specifically, detection method includes:

Differentiate: (1) takes this preparation or its content 3g, adds ethanol 10mL, close plug, soaks 20 minutes, shake constantly, filter Crossing, filtrate is as need testing solution；Separately take cinnamic aldehyde reference substance, add ethanol and make every 1mL solution containing 1 μ L, as reference substance Solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw need testing solution 10～15 μ L, comparison Product solution 2 μ L, puts on same silica gel g thin-layer plate, with petroleum ether-ethyl acetate=17:3 that boiling range is 60～90 DEG C for launching Agent, launches, and takes out, dries, and spray is with dinitrophenylhydrazine ethanol test solution；In test sample chromatograph, in position corresponding with reference substance chromatograph Put, the speckle of aobvious same color；

(2) take this preparation or its content 4g, add methanol 10mL, supersound process 30 minutes, filter, take filtrate as examination Product solution；Separately take dehydrocostuslactone reference substance, costunolide reference substance, add methanol and be respectively prepared molten containing 0.5mg of every 1mL Liquid, as reference substance solution；According to one the annex VI B thin layer chromatography test of Chinese Pharmacopoeia version in 2010, draw above-mentioned three kinds molten The each 5 μ L of liquid, put respectively on same silica gel g thin-layer plate, with the upper solution of cyclohexane-carboxylic acid ethyl ester-formic acid=15:5:1 are Developing solvent, launches, and takes out, dries, and spray, with 1% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, On position corresponding with reference substance chromatograph, the speckle of aobvious same color；

(3) taking this preparation or its content 3g, add ethyl acetate 10mL, supersound process 15 minutes, filter, filtrate is evaporated, Residue adds dehydrated alcohol 2mL makes dissolving, as need testing solution；Separately take ursolic acid reference substance, add dehydrated alcohol and make every 1mL and contain The solution of 1mg, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw above-mentioned Two kinds of each 5 μ L of solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20:4:0.5 for launching Agent, launches, and takes out, dries, and spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear；Test sample chromatograph In, on position corresponding with reference substance chromatograph, aobvious identical aubergine speckle；

(4) take this preparation or its content 3g, put in tool plug centrifuge tube, add the petroleum ether 10mL that boiling range is 30～60 DEG C, Supersound process 20 minutes, filters, and filtrate volatilizes, and residue adds dehydrated alcohol 1mL makes dissolving, as need testing solution；Separately take lucky horse Ketone reference substance, adds dehydrated alcohol and makes every 1mL solution containing 0.4mg, as reference substance solution；According to Chinese Pharmacopoeia version one in 2010 Portion's annex VI B thin layer chromatography is tested, and draws each 10 μ L of above two solution, puts respectively on same silica gel g thin-layer plate, with boiling Journey be the petroleum ether-acetone and ethyl acetate=94:5:1 of 30～60 DEG C be developing solvent, launch, take out, dry, spray with 1% Rhizoma et radix valerianae Aldehyde sulfuric acid solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, on position corresponding with reference substance chromatograph, The speckle of aobvious same color；

(5) taking this preparation or its content 4g, add diethyl ether 50mL, supersound process 10 minutes, filters, and filtrate is evaporated, residue Add methanol 1mL and make dissolving, as need testing solution；Separately take Poria control medicinal material lg, be made in the same way of control medicinal material solution；According to China Pharmacopeia one annex VI B thin layer chromatography test of version in 2010, draws each 2 μ L of above two solution, puts respectively in same silica gel G On lamellae, with toluene-ethyl acetate-formic acid=20:5:0.5 as developing solvent, launching, take out, dry, spray is with 2% vanillin Sulfuric acid solution-ethanol=4:1 mixed solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, with reference substance On the corresponding position of chromatograph, the principal spot of aobvious same color；

(6) taking this preparation or its content 4g, add diethyl ether l0mL, close plug, shakes 10 minutes, filters, and filtrate volatilizes, residue Add acetone 2mL and make dissolving, as need testing solution；Separately take paeonol reference substance, add acetone and make every 1mL solution work containing 2mg For reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw above two solution each 10 μ L, puts respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate-glacial acetic acid=4:1:0.1 as developing solvent, launches, Taking out, dry, spray, with 2% vanillin-sulfuric acid ethanol solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, On position corresponding with reference substance chromatograph, the speckle of aobvious same color；

(7) taking this preparation or its content 3g, add ethanol 10mL, shake 5 minutes, filter, filtrate is evaporated, and residue adds ethanol 2mL makes dissolving, as need testing solution；Separately take peoniflorin reference substance, add ethanol and make every 1mL solution containing 2mg, as comparison Product solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw each 4 μ L of above two solution, respectively Point is on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methyl alcohol-formic acid=40:5:10:0.2 as developing solvent, launches, takes Going out, dry, spray, with 5% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, with reference substance chromatograph On corresponding position, aobvious identical bluish violet speckle；

Assay: (1) Radix Paeoniae Rubra is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol- 0.05mol/L potassium dihydrogen phosphate=40:65 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 230nm, number of theoretical plate should be not less than 3000 based on peoniflorin peak；

It is appropriate that the preparation of reference substance solution takes peoniflorin reference substance, accurately weighed, adds methanol and makes every 1mL containing peoniflorin The solution of 30 μ g, to obtain final product；

The preparation of need testing solution takes this preparation or its content 1g, accurately weighed, to 50mL conical flask, adds first Alcohol extraction solvent about 25mL, supersound extraction 10min, take out, filter, take subsequent filtrate, to obtain final product；

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

(2) Herba Polygoni cymosi is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol-0.4% Phosphoric acid solution=50:50 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 360nm；Number of theoretical plate presses Cortex querci dentatae Element peak meter should be not less than 3000；

It is appropriate that the preparation of reference substance solution takes Quercetin reference substance, accurately weighed, adds methanol and makes every 1mL containing Quercetin The solution of 30 μ g, to obtain final product；

The preparation of need testing solution takes this preparation or its content 2g, accurately weighed, to round-bottomed flask, and addition hydrochloric acid: The mixed solution 50mL of methanol=1:4, weighed weight, put 90 DEG C of water-bath reflux, extract, 1 hour, take out, cooling, weighed weight, Supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；

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

In order to ensure the science, reasonable, feasible of the present invention program, by carrying out series of experimental research and investigation, thus Determine the solution of the present invention.In the present invention with treatment endometriosis Chinese medicine granules as object of study.

1 character

The experimental result of the middle test agent according to three batches is drafted: this preparation is yellowish-brown granule；Feeble QI, mildly bitter flavor.

2 differentiate

2.1 material

Electronics analytical balance (METTLER TOLEDO AB204-S)；HH-4 digital display thermostat water bath (China of Changzhou Australia Instrument Ltd.)；The infrared Quick drying box of YHG-600-S-II type (He De Shi Qi company limited)；Ultrasonic cleaner is (peaceful Bo Xinzhi bio tech ltd)；DHG9070A-electric drying oven with forced convection (above Nereid's grand experimental facilities company limited)；ZF Ultraviolet analysis instrument for three purposed；Thin layer chromatography silica gel is chemical pure, and reagent is analytical pure.

Reagent peoniflorin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110736-200526)；Paeonol pair According to product (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110021-200408)；Cinnamic aldehyde reference substance (China's pharmaceutical biological product Calibrating institute, lot number 110175-200826)；Dehydrocostuslactone reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 100341-200302)；Costunolide reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110711-200407)；Bears Fruit acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110329-200611)；(China's medicine is raw for 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-reference substance Tetramune examines and determine institute, lot number 110003-200521)；Poria control medicinal material (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 100611-200621)；Chinese medicine granules (benefit one hundred pharmaceutical factory's pilot scale center, Guizhou, the lot number for the treatment of endometriosis 20131001、20131002、20131003)；Chloroform, methanol, formic acid, hexamethylene, ethyl acetate, glacial acetic acid, acetone, oil Ether, ethanol etc. are analytical pure.

2.2 qualitative examination method and results

This experiment of thin-layer identification method of Radix Paeoniae Rubra is with reference to the indentification by TLC of " Chinese Pharmacopoeia " 2010 editions middle Radix Paeoniae Rubra Method, through test of many times, method is sensitive, and repeatability and specificity are good, facilitate feasible, and negative noiseless, result is shown in Fig. 1.

This experiment of thin-layer identification method of Cortex Moutan is with reference to the thin layer chromatography of " Chinese Pharmacopoeia " 2010 editions middle Cortex Moutans Discrimination method, through test of many times, method is sensitive, and repeatability and specificity are good, facilitate feasible, and negative noiseless, result is shown in Fig. 2.

This experiment of thin-layer identification method of Ramulus Cinnamomi is with reference to the indentification by TLC of " Chinese Pharmacopoeia " 2010 editions middle Ramulus Cinnamomi Method, through test of many times, method is sensitive, and repeatability and specificity are good, facilitate feasible, and negative noiseless, result is shown in Fig. 3.

This experiment of thin-layer identification method of the Radix Aucklandiae is with reference to the indentification by TLC of " Chinese Pharmacopoeia " 2010 editions middle Radix Aucklandiae Method, through test of many times, method is sensitive, and repeatability and specificity are good, facilitate feasible, and negative noiseless, result is shown in Fig. 4.

This experiment of thin-layer identification method of Fructus Corni is with reference to the thin layer chromatography of " Chinese Pharmacopoeia " 2010 editions middle Fructus Corni Discrimination method, through test of many times, method is sensitive, and repeatability and specificity are good, facilitate feasible, and negative noiseless, result is shown in Fig. 5.

This experiment of thin-layer identification method of Rhizoma Curcumae is with reference to the indentification by TLC of " Chinese Pharmacopoeia " 2010 editions middle Rhizoma Curcumae Method, through test of many times, method is sensitive, and repeatability and specificity are good, facilitate feasible, and negative noiseless, result is shown in Fig. 6.

This experiment of thin-layer identification method of Poria is with reference to the indentification by TLC of " Chinese Pharmacopoeia " 2010 editions middle Poria Method, through test of many times, method is sensitive, and repeatability and specificity are good, facilitate feasible, and negative noiseless, result is shown in Fig. 7.

3 check

With reference to the regulation under the Pharmacopoeia of the People's Republic of China one annex I C granule item of version in 2010, to this preparation Granularity, moisture, melting, content uniformity, microbial limit etc. investigated.

3.1 granularity inspections

Granulometry is with reference to Pharmacopoeia of the People's Republic of China version in 2010 second mensuration of one annex XI B.Take this Granule 10 bags, weighed weight, to sieve in putting the medicine sieve of regulation, keep level, left and right comes and goes and sieves 3～5min gently. Take and can not calculate percentage by a sieve and the weighed weight of summation that No. five sieves can be passed through.The sample of three batches after testing Product are respectively less than 15%, meet regulation.The results are shown in Table 1.

Table 1 three batch sample granularity checks

3.2 content uniformity inspections

Taking this granule 10 bags, respectively the weight of weighed every bag of content, every packed amount, compared with labelled amount, must not surpass Cross labelled amount ± 10%, i.e. 4.50～5.50g.The sample of three batches all meets regulation after measured.The results are shown in Table 2.

Table 2 three batch sample content uniformity checks

3.3 melting

Taking this granule test sample 10g, add hot water 20 times, stir 5min, observe immediately, sol particle should all be melted Change, it is allowed to having slight haze, three batch test samples all meet regulation after measured.The results are shown in Table 3.

Table 3 three batch sample melting checks

3.4 moisture inspections

Moisture inspection technique measures with reference to the Pharmacopoeia of the People's Republic of China one annex IX H the first method of version in 2010, takes this Preparation 4g, accurately weighed, it is laid in and is dried to the flat weighing botle of constant weight, thickness is less than 5mm, accurately weighed, opens bottle Cover at 100～105 DEG C of dry 5h, bottle cap is built, move in exsiccator, cool down 30min, accurately weighed, then at said temperature It is dried 1h, cooling, weighs, to the double difference weighed less than 5mg, according to the weight of less loss, calculate test sample Middle water content.Standard regulation moisture is less than 6.0%, and three batch sample are respectively less than 6.0% after measured, meet regulation.Result is shown in Table 4.

Table 4 three batch sample moisture checks

3.5 heavy metal inspections

Measure according to lead, cadmium, arsenic, hydrargyrum, copper algoscopy (one annex Ⅸ B of " Chinese Pharmacopoeia " version in 2010).In surveyed data Lead, arsenic, copper all not less than 2ppm, cadmium and hydrargyrum all not less than 0.2ppm.The results are shown in Table 5.

Table 5 heavy metal and harmful element measurement result table

3.6 microbial limit

Check according to microbial limit test (one annex Ⅹ III C of " Chinese Pharmacopoeia " version in 2010), the three micro-lifes of batch sample Thing limit test, all meets regulation.The results are shown in Table 6.

Table 6 three batch sample limit test of microbe result table

4 assays

4.1 instruments and reagent

Instrument Agilent1100 high performance liquid chromatograph, ultrasonic washing unit (HS20500D), balance (AB204-S), Buchi Rotary Evaporators, HH-4 digital display thermostat water bath (Changzhou Ao Hua Instrument Ltd.).

Reagent peoniflorin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110736-200526)；Quercetin compares Product (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 100081-200406)；The Chinese medicine granules for the treatment of endometriosis (benefit one hundred pharmaceutical factory's pilot scale center, Guizhou, lot number 20131001,20131002,20131003)；Methanol (chromatographically pure and analytical pure)； Hydrochloric acid；Phosphoric acid；Potassium dihydrogen phosphate；Wahaha Pure Water etc..

The assay of 4.2 Radix Paeoniae Rubra

The preparation of need testing solution:

(1) investigation of different solvents

Take the granule 1g under content uniformity item, accurately weighed, to 50mL conical flask, it is separately added into methanol and ethanol Extraction solvent about 25mL, weighed weight, supersound process (power 250W, frequency 20HZ) 20min, takes out, weighed weight, uses methanol With the weight that less loss supplied by ethanol.Filter, take subsequent filtrate, to obtain final product.The test sample that precision absorption different solvents extracts respectively again is each 10 μ L, measure chromatographic condition by paeoniflorin content under " Chinese Pharmacopoeia " version Radix Paeoniae Rubra item in 2010, inject high performance liquid chromatograph, survey Fixed, to obtain final product.Result is as shown in Figure 8 and Figure 9.

Conclusion: in view of the chemical property of peoniflorin, the confession measured with reference to peoniflorin in 2010 editions " Chinese Pharmacopoeia " Radix Paeoniae Rubra Test product processing method is investigated on the basis of Extraction solvent with methanol and the ethanol different solvents shadow to the extraction ratio of peoniflorin Ringing, under the same conditions, the test sample index components peak area with ethanol as Extraction solvent supplies for Extraction solvent less than with methanol Test product index components peak area, and the test sample index components peak with ethanol as Extraction solvent is not kept completely separate, and illustrates with second Alcohol be Extraction solvent test sample in peoniflorin be not fully extracted separation, therefore select molten using methanol solution as optimum extraction Agent.

(2) investigation of extracting mode

1. the granule 1g under supersound extraction takes content uniformity item is accurately weighed, to 50mL conical flask, adds methanol and carries Take solvent about 25mL, weighed weight, supersound process (power 250W, frequency 20HZ) 20min, take out, weighed weight, mend with methanol The weight of foot less loss.Filter, take subsequent filtrate, to obtain final product.

2. the granule 1g under reflux, extract, takes content uniformity item is accurately weighed, to 50mL round-bottomed flask, adds methanol Extraction solvent about 25mL, weighed weight.80 DEG C of reflux, extract, 30min of water-bath, take out, cooling, and weighed weight is supplied with methanol and subtracted The weight lost.Filter, take subsequent filtrate, to obtain final product.

3. shake the granule 1g taken under content uniformity item accurately weighed, to 50mL conical flask, add methanol and carry Take solvent about 25mL, shake 20min.Filter, take subsequent filtrate, to obtain final product.

Precision draws test sample and each 10 μ L of peoniflorin reference substance of supersound extraction and reflux, extract, respectively, by " middle traditional Chinese medicines Allusion quotation " paeoniflorin content measures chromatographic condition under 2010 years version Radix Paeoniae Rubra items, and inject high performance liquid chromatograph, measure, to obtain final product.Result is such as Shown in Figure 10.

Conclusion: understood supersound extraction and reflux, extract, by above-mentioned chromatogram, shaked peoniflorin peak area in extraction chromatography figure Without large change, illustrate that above-mentioned three kinds of extracting method all can extract peoniflorin, it is contemplated that the popularity rate of Modern Laboratory instrument With cost-effective, the time, therefore select ultrasonic extracting method.

(3) ultrasonic time is investigated

Take the granule 1g under content uniformity item accurately weighed, to 50mL conical flask, add methanol extraction solvent about 25mL.Weighed weight, respectively supersound process (power 250W, frequency 20HZ) 10min, 20min, 30min, take out, weighed weight, The weight of less loss is supplied with methanol.Filter, take subsequent filtrate, to obtain final product.

Respectively precision draw supersound extraction 10,20, each 10 μ L of test sample of 30min, red by " Chinese Pharmacopoeia " version in 2010 Under Chinese herbaceous peony item, paeoniflorin content measures chromatographic condition, injects high performance liquid chromatograph, measures, to obtain final product.Result is as shown in figure 11.

Conclusion: by above-mentioned chromatogram understand ultrasonic time 10,20,30min peoniflorin peak area is without large change.Therefore it is Saving time and power consumption, the method for selecting ultrasonic 10min.

In sum, the preparation method of need testing solution is: take the granule 1g under content uniformity item accurately weighed, extremely In 50mL conical flask, add methanol extraction solvent about 25mL, weighed weight, supersound process (power 250W, frequency 20HZ) 10min, takes out, weighed weight, supplies the weight of less loss with methanol.Filter, take subsequent filtrate, to obtain final product.

Chromatographic condition is preferred:

(1) chromatographic column is preferred

The present invention uses DIKMA post respectively, and agela post, Agilent post are analyzed, and compare separating resulting, three kinds of chromatographic columns Comparing, peak shape has different, and retention time differs greatly, and the peak shape of agela post is preferable, separating effect agela post ratio DIKMA post, Agilent post are good, therefore determine that agela post is as assay analytical column.Result is shown in Figure 12.

(2) chromatograph wavelength is preferred

When carrying out paeoniflorin content and measuring chromatography condition, with reference to Pharmacopoeia of the People's Republic of China version in 2010 Under one Radix Paeoniae Rubra item, the assay chromatographic condition of peoniflorin, has carried out the screening of chromatograph wavelength.Be respectively compared 220nm, The separating degree of peoniflorin, theoretical cam curve and its peak area under 230nm, 235nm, 240nm equiwavelength, result proves at wavelength At 230nm, the separating degree of peoniflorin is best, and peak area is maximum, and interference factor is minimum.Result is shown in Figure 13.

(3) chromatographic column temperature is preferred

The present invention compares 25 DEG C, 30 DEG C, the column temperature of the 35 DEG C impact on peoniflorin, test result indicate that the change of column temperature Have also been changed the retention time of peoniflorin, and under the conditions of 25 DEG C of column temperatures, peoniflorin peak separating degree is best, theoretical cam curve is high. Result is shown in Figure 14.

(4) flowing is mutually preferred

The present invention with reference to the chromatographic condition of the assay of peoniflorin under one Radix Paeoniae Rubra item of " Chinese Pharmacopoeia " version in 2010, Compare acetonitrile-0.05mol/L potassium dihydrogen phosphate=40:65, methanol-0.05mol/L potassium dihydrogen phosphate=35: 70, methanol-0.05mol/L potassium dihydrogen phosphate=40:65 and methanol-0.05mol/L potassium dihydrogen phosphate=45:60 Flowing phase, show that peoniflorin descends separating degree best in the flowing of methanol-0.05mol/L potassium dihydrogen phosphate=40:65 mutually, reason The opinion number of plates is the highest.Result is shown in Figure 15.

In sum, determine that optimal chromatographic condition is: with methanol-0.05mol/L potassium dihydrogen phosphate=40:65 be Flowing phase, flow velocity 1.0mL/min, detection wavelength is 230nm, column temperature 25 DEG C.

Specificity is tested:

Use HPLC-array diode detector, by the chromatographic condition drafted, draw reference substance solution and test sample respectively Solution is measured, and detection wavelength is 230nm.Measurement result shows, protects with peoniflorin reference substance chromatographic peak in need testing solution The chromatographic peak ultraviolet spectrogram staying time consistency is consistent with reference substance ultraviolet spectrogram, test compound in need testing solution chromatograph Peak purity is more than 99%, and chromatographic condition determined by explanation can have preferably separation to above-claimed cpd in test sample, is built Vertical method has good specificity.Result is shown in Figure 16, Figure 17, Figure 18 and Figure 19.

Linear relationship:

It is appropriate that precision weighs peoniflorin reference substance, adds methanol and dissolves that to make the reference substance that mass concentration is 257 μ g/mL molten Liquid, injects high performance liquid chromatograph by the chromatographic condition drafted and measures.Peoniflorin sample size be respectively 1 μ L, 2.5 μ L, 5 μ L, 7.5 μL、12.5μL.With peak area, sample introduction quality is carried out linear regression, obtain regression equation, correlation coefficient and the line of each tested composition Property scope.Result such as table 7 and Figure 20: peoniflorin mass concentration is good linear relation at 0.26～3.21 μ g range.

Table 7

Stability test:

Preparation need testing solution 1 part is prepared, with the chromatographic condition drafted, by 0h, 2h, 4h, 8h, 12h according to the method drafted Sample introduction 10 μ L (20131102) respectively, measures peoniflorin Component peak area.Result is stable in showing need testing solution 12h.Its RSD It is 1.80%, meets quality standard technology and require (RSD is within 3%), have good stability.The results are shown in Table 8.

Table 8 peoniflorin stability result

Precision test:

Preparation need testing solution 1 part is prepared, with the chromatographic condition drafted, continuous sample introduction 6 times according to the method drafted (20131102), each 10 μ L, measure peoniflorin Component peak area, 6 peak area RSD are 0.78% as a result, meet quality mark Quasi-technology requires (RSD is within 3%), and precision is good.The results are shown in Table 9.

Table 9 peoniflorin precision result

Replica test:

Take the Chinese medicine granules of the treatment endometriosis of same lot number (20131102), prepare according to the method drafted Preparation need testing solution 6 parts, with the chromatographic condition drafted, each 10 μ L, measures peoniflorin Component peak area, parameter respectively Content, RSD is 2.03%, meets quality standard technology and requires (RSD is all within 3%), and repeatability is good.The results are shown in Table 10.

Table 10 peoniflorin repeatability result

Recovery test:

Use average recovery method, take (20131102) 9 parts of the Chinese medicine granules sample for the treatment of endometriosis, often Part 0.5g, accurately weighed, by the method drafted, sample is processed, adds appropriate peoniflorin reference substance solution, make low, Middle and high 3 kinds of variable concentrations need testing solutions.By the chromatographic condition drafted, it is measured, is calculated as follows out the response rate, calculate Formula is as follows:

The response rate=(C-A)/B × 100%

A: containing the amount (mg) of corresponding reference substance in sample

B: reference substance addition (mg)

C: measured amount (mg)

Table 11 peoniflorin sample-adding reclaims result

Result is as shown in table 11: the average recovery rate of this sample is 99.29%, and RSD is 1.73%, and accuracy is good.

The assay of 4.3 Herba Polygoni cymosis

The preparation of need testing solution:

(1) investigation of different solvents

Take the granule 2g under content uniformity item accurately weighed, to 100mL round-bottomed flask, be separately added into hydrochloric acid: methanol (v:v)=1:4 and hydrochloric acid: the Extraction solvent about 50mL of ethanol (v:v)=1:4, weighed weight.90 DEG C of reflux, extract, 1 of water-bath are little Time, take out, cooling, weighed weight, supply the weight of less loss with corresponding solvent.Filter, take subsequent filtrate, to obtain final product.The most smart again The test sample that close absorption different solvents extracts and each 10 μ L of Quercetin reference substance, by under " Chinese Pharmacopoeia " version Herba Polygoni cymosi item in 2010 Quercetin content measures chromatographic condition, injects high performance liquid chromatograph, measures, to obtain final product.Result is as shown in figure 21 and figure.

Conclusion: in view of the chemical property of Quercetin, measure with reference to Quercetin in 2010 editions " Chinese Pharmacopoeia " Herba Polygoni cymosis Test sample processing method is investigated with hydrochloric acid on the basis of Extraction solvent: the mixed solution of methanol (v:v)=1:4 and hydrochloric acid: The impact on the extraction ratio of Quercetin of the mixed solution different solvents of ethanol (v:v)=1:4, under the same conditions, with hydrochloric acid: second The test sample peak area that mixed solution is Extraction solvent of alcohol (v:v)=1:4 is less than with hydrochloric acid: the mixing of methanol (v:v)=1:4 Solution is Extraction solvent test sample peak area (peak area ratio is 362:175), illustrates with hydrochloric acid: the mixing of ethanol (v:v)=1:4 Solution be Extraction solvent test sample in Quercetin be not fully extracted, therefore select with hydrochloric acid: methanol (v:v)=1:4's is mixed Close solution as optimum extraction solvent.

(2) investigation of different extracting modes

1. the granule 2g under supersound extraction takes content uniformity item is accurately weighed, to 100mL volumetric flask, and addition hydrochloric acid: The mixed solution about 50mL of methanol (v:v)=1:4, weighed weight, supersound process (power 250W, frequency 20HZ) 30min, take Go out, weighed weight, supply the weight of less loss with methanol.Filter, take subsequent filtrate, to obtain final product.

2. the granule 2g under reflux, extract, takes content uniformity item is accurately weighed, to 100mL round-bottomed flask, adds salt Acid: the Extraction solvent about 50mL of methanol (v:v)=1:4, weighed weight.90 DEG C of reflux, extract, of water-bath 1 hour, take out, and cooling claims Determine weight, supply the weight of less loss with methanol.Filter, take subsequent filtrate, to obtain final product.

Precision draws test sample and each 10 μ L of Quercetin reference substance of supersound extraction and reflux, extract, respectively, by " middle traditional Chinese medicines Allusion quotation " quercetin content measures chromatographic condition under 2010 years version Herba Polygoni cymosi items, and inject high performance liquid chromatograph, measure, to obtain final product.Result As shown in figure 23.

Conclusion: understood supersound extraction chromatogram by above-mentioned chromatogram and there is no the appearance of Quercetin peak, illustrate that ultrasonic method does not has Extract Quercetin, therefore select reflux extraction method.

(3) reflux extracting time is investigated

Take the granule 2g under content uniformity item accurately weighed, to 100mL round-bottomed flask, addition hydrochloric acid: methanol (v:v) The Extraction solvent of=1:4 about 50mL, weighed weight.Water-bath 90 DEG C of reflux, extract, 0.5h, 1h, 1.5h respectively, take out, and cooling claims Determine weight, supply the weight of less loss with methanol.Filter, take subsequent filtrate, to obtain final product.

Precision draws test sample and each 10 μ L of Quercetin reference substance of reflux, extract, 0.5h, 1h, 1.5h respectively, by " China Pharmacopeia " quercetin content measures chromatographic condition under 2010 years version Herba Polygoni cymosi items, and inject high performance liquid chromatograph, measure, to obtain final product.Knot Fruit is as shown in figure 24.

Conclusion: by above-mentioned chromatogram understand extraction time be 0.5 hour Quercetin peak area less than 1 hour extraction time and The Quercetin peak area of 1.5 hours, and the Quercetin peak area of 1 hour extraction time and 1.5 hours has certain difference, during extraction Between be the Quercetin peak area of the 1.5 hours Quercetin peak area that is approximately less than 1 hour extraction time, after this explanation is extracted 1 hour Quercetin is the most extracted completely, and the time is oversize on the contrary according to the loss becoming Quercetin.Therefore to the time of saving and power consumption, select The method extracting 1 hour.

In sum, the preparation method of need testing solution is: take the granule 2g under content uniformity item accurately weighed, extremely In 100mL round-bottomed flask, addition hydrochloric acid: the Extraction solvent about 50mL of methanol (v:v)=1:4, weighed weight.Water-bath 90 DEG C respectively Reflux, extract, 1 hour, takes out, cooling, and weighed weight supplies the weight of less loss with methanol.Filter, take subsequent filtrate, to obtain final product.Chromatograph Condition is preferred:

(1) chromatographic column is preferred

The present invention uses DIKMA post respectively, and Agela post Agilent post is analyzed, and compares separating resulting, three kinds of chromatographic columns Comparing, peak shape has different, and retention time differs greatly, and the peak shape of Agela post is preferable, separating effect Agela post ratio DIKMA post, Agilent post are good, therefore determine that Agela post is as assay analytical column.Result is as shown in figure 25.

(2) chromatograph wavelength is preferred

When carrying out quercetin content and measuring chromatography condition, with reference to Pharmacopoeia of the People's Republic of China version in 2010 Under one Herba Polygoni cymosi item, the assay chromatographic condition of Quercetin, has carried out the screening of chromatograph wavelength.Be respectively compared 340nm, The separating degree of Quercetin, theoretical cam curve and its peak area under 350nm, 355nm, 360nm equiwavelength, result proves at wavelength At 360nm, the separating degree of Quercetin is best, and peak area is maximum, and interference factor is minimum.Result is as shown in figure 26.

(3) chromatographic column temperature is preferred

The present invention compares 25 DEG C, 30 DEG C, the column temperature of the 35 DEG C impact on Quercetin, test result indicate that 30 DEG C of column temperature bars Under part, separating degree is best, and retention time is short, and theoretical cam curve is high.Result is as shown in figure 27.

(4) flowing is mutually preferred

The present invention with reference to measure containing of Quercetin under the Pharmacopoeia of the People's Republic of China one Herba Polygoni cymosi item of version in 2010 Fixed chromatographic condition, compares according to volume ratio, methanol-0.4% phosphoric acid water=55:45, methanol-0.4% phosphoric acid water=50: 50, the flowing phase of methanol-0.4% phosphoric acid water=45:55 and acetonitrile-0.4% phosphoric acid water=50:50, show that Quercetin is in first The flowing of alcohol-0.4% phosphoric acid water=50:50 descends separating degree best mutually, therefore determines that flowing is mutually for methanol-0.4% phosphoric acid water=50: 50.Result is as shown in figure 28.

In sum, determine that optimal chromatographic condition is: methanol-0.4% phosphoric acid water=50:50 is flowing phase, flow velocity 1.0mL/min, detection wavelength is 360nm, column temperature 30 DEG C.

Specificity is tested:

Use HPLC-array diode detector, by the chromatographic condition drafted, draw reference substance solution and test sample respectively Solution is measured, and detection wavelength is 360nm.Measurement result shows, protects with Quercetin reference substance chromatographic peak in need testing solution The chromatographic peak ultraviolet spectrogram staying time consistency is consistent with reference substance ultraviolet spectrogram, test compound in need testing solution chromatograph Peak purity is more than 99%, and chromatographic condition determined by explanation can have preferably separation to above-claimed cpd in test sample, is built Vertical method has good specificity.Result is as shown in Figure 29, Figure 30, Figure 31 and Figure 32.

Linear relationship:

It is appropriate that precision weighs Quercetin reference substance, adds methanol dissolving and makes the reference substance solution that mass concentration is 90 μ g/mL, Inject high performance liquid chromatograph by the chromatographic condition drafted to measure.Quercetin sample size is respectively 0.1 μ L, 0.3 μ L, 0.5 μ L, 1 μ L、2μL.With peak area, sample introduction quality is carried out linear regression, obtain the regression equation of tested composition, correlation coefficient and the range of linearity. Result is as shown in table 12 and Figure 33: Quercetin mass concentration is good linear relation at 0.009～0.180 μ g range.

Table 12

Stability test:

Preparation need testing solution 1 part is prepared, with the chromatographic condition drafted, by 0h, 2h, 4h, 8h, 12h according to the method drafted Sample introduction 10 μ L (20131102) respectively, measures quercetin component peak area.Result is stable in showing need testing solution 12h.Its RSD It is 1.60%, meets quality standard technology and require (RSD is within 3%), have good stability.The results are shown in Table 13.

Table 13 Quercetin stability result

Precision test:

Preparation need testing solution 1 part is prepared, with the chromatographic condition drafted, continuous sample introduction 6 times according to the method drafted (20131102), each 10 μ L, measure quercetin component peak area, 6 peak area RSD are 0.82% as a result, meet quality mark Quasi-technology requires (RSD is within 3%), and precision is good.The results are shown in Table 14.

Table 14 Quercetin precision result

Replica test:

Take the Chinese medicine granules of the treatment endometriosis of same lot number (20131102), according to the method drafted respectively Prepare preparation need testing solution 6 parts, with the chromatographic condition drafted, each 10 μ L, measure quercetin component peak area respectively, calculate Index content, RSD is 1.81%, meets quality standard technology and requires (RSD is all within 3%), and repeatability is good.The results are shown in Table 15。

Table 15 Quercetin repeatability result

Recovery test:

Use average recovery method, take (20131102) 9 parts of the Chinese medicine granules sample for the treatment of endometriosis, often Part 1g, accurately weighed, by the sample solution preparation method drafted, sample is processed, add appropriate Quercetin reference substance molten Liquid, makes basic, normal, high 3 kinds of variable concentrations need testing solutions.It is measured by the chromatographic condition drafted, is calculated as follows out back Yield, calculating formula is as follows:

The response rate=(C-A)/B × 100%

A: containing the amount (mg) of corresponding reference substance in sample

B: reference substance addition (mg)

C: measured amount (mg)

Table 16 sample-adding reclaims result

Result is as shown in table 16: the average recovery rate of this sample is 99.88%, and RSD is 2.19%, and accuracy is good.

4.4 assay results

Take the Chinese medicine granules of the treatment endometriosis of 3 batches respectively, by need testing solution preparation method and The chromatographic condition drafted, measures Quercetin and the content of peoniflorin composition, and measurement result is shown in Table 17.

Chinese medicine granules assay result (mg/g) of endometriosis treated by table 17

Above-mentioned 3 batch samples record Quercetin and are respectively 0.1258mg/g and 2.9475mg/g with peoniflorin average content.From Large-scale production considers in high volume, with 80% calculating of every gram of this preparation Quercetin with the content limit average content of peoniflorin, i.e. Quercetin 0.1258 × 5 × 0.8=0.504mg/ bag, peoniflorin 2.9475 × 5 × 0.8=11.79mg/ bag, tentatively specify this Every bag of preparation (5g) contains Radix Paeoniae Rubra with peoniflorin (C₂₃H₂₈O₁₁) meter, 11.8mg must not be less than；Every bag of this preparation contains Herba Polygoni cymosi with Cortex querci dentatae Element (C₁₅H₁₀O₇) meter, 0.5mg must not be less than.

The invention have benefit that: the detection method of the present invention is by Ramulus Cinnamomi, Radix Paeoniae Rubra, the Radix Aucklandiae, Paeonia suffruticosa in preparation Skin, Fructus Corni, Poria, seven medical materials of Rhizoma Curcumae differentiate, acquired results feminine gender is noiseless, and specificity is relatively strong, with reference substance Or the corresponding position of control medicinal material shows the speckle of same color；Quercetin in said preparation and paeoniflorin content are measured, precision, Stability, repeatability, system suitability and average recovery all meet the requirements.The detection method of this preparation is easy, quickly, can Leaning on, accurately, specificity is strong, and precision is high, favorable reproducibility, and the response rate is high, and measurement result is accurate, can effectively control drug quality, So that it is guaranteed that the stable and clinical application of product quality is safely, effectively.

Accompanying drawing explanation

Fig. 1 is Radix Paeoniae Rubra thin layer discriminating figure；

Fig. 2 is Cortex Moutan thin layer discriminating figure；

Fig. 3 is Ramulus Cinnamomi thin layer discriminating figure；

Fig. 4 is Radix Aucklandiae thin layer discriminating figure；

Fig. 5 is Fructus Corni thin layer discriminating figure；

Fig. 6 is Rhizoma Curcumae thin layer discriminating figure；

Fig. 7 is Poria thin layer discriminating figure；

Fig. 8 be Radix Paeoniae Rubra assay in different solvents extract contrast color spectrogram；

Fig. 9 be Radix Paeoniae Rubra assay in peoniflorin reference substance chromatogram；

Figure 10 be Radix Paeoniae Rubra assay in extracting method contrast color spectrogram；

Figure 11 be Radix Paeoniae Rubra assay in ultrasonic time contrast color spectrogram；

Figure 12 be Radix Paeoniae Rubra assay in different chromatographic column contrast color spectrograms；

Figure 13 be Radix Paeoniae Rubra assay in different wave length contrast color spectrogram；

Figure 14 be Radix Paeoniae Rubra assay in different column temperature contrast color spectrograms；

Figure 15 be Radix Paeoniae Rubra assay in different flowing relative colorimetric's spectrograms；

Figure 16 be Radix Paeoniae Rubra assay in treat the Chinese medicine granules sample chromatogram figure of endometriosis；

Figure 17 be Radix Paeoniae Rubra assay in peoniflorin reference substance chromatogram；

Figure 18 be Radix Paeoniae Rubra assay in lack Radix Paeoniae Rubra negative sample chromatogram；

Figure 19 be Radix Paeoniae Rubra assay in solvent chromatogram；

Figure 20 be Radix Paeoniae Rubra assay in peoniflorin canonical plotting；

Figure 21 be Herba Polygoni cymosi assay in different solvents extract contrast color spectrogram；

Figure 22 be Herba Polygoni cymosi assay in Quercetin reference substance chromatogram；

Figure 23 be Herba Polygoni cymosi assay in extracting method contrast color spectrogram；

Figure 24 be Herba Polygoni cymosi assay in extraction time contrast color spectrogram；

Figure 25 be Herba Polygoni cymosi assay in different chromatographic column contrast color spectrograms；

Figure 26 be Herba Polygoni cymosi assay in different wave length contrast color spectrogram；

Figure 27 be Herba Polygoni cymosi assay in different column temperature contrast color spectrograms；

Figure 28 be Herba Polygoni cymosi assay in different flowing relative colorimetric's spectrograms；

Figure 29 be Herba Polygoni cymosi assay in treat the Chinese medicine granules sample chromatogram figure of endometriosis；

Figure 30 be Herba Polygoni cymosi assay in Quercetin reference substance chromatogram；

Figure 31 be Herba Polygoni cymosi assay in lack Herba Polygoni cymosi negative sample chromatogram；

Figure 32 be Herba Polygoni cymosi assay in solvent chromatogram；

Figure 33 be Herba Polygoni cymosi assay in Quercetin canonical plotting；

The implication of reference in figure: in Fig. 1: 1 is peoniflorin reference substance, 2 for lacking Radix Paeoniae Rubra negative control, and 3,4,5 respectively For treating the Chinese medicine granules test sample 20131001,20131002,20131003 of endometriosis；In Fig. 2: 1 is scarce Cortex Moutan negative control, 2 is paeonol reference substance, and 3,4,5 are respectively the Chinese medicine granules for the treatment of endometriosis for examination Product 20131001,20131002,20131003；In Fig. 3: 1 is cinnamic aldehyde reference substance, 2 for lacking Ramulus Cinnamomi negative control, 3,4,5 points Wei not treat the Chinese medicine granules test sample 20131001,20131002,20131003 of endometriosis；In Fig. 4: 1 is Lacking Radix Aucklandiae negative control, 2 is dehydrocostuslactone reference substance, and 3 is costunolide reference substance, and 4,5,6 respectively treat uterus The Chinese medicine granules test sample 20131001,20131002,20131003 of endometrium ectopia；In Fig. 5: 1 is ursolic acid reference substance, 2 for lack Fructus Corni negative control, 3,4,5 be respectively treatment endometriosis Chinese medicine granules test sample 20131001, 20131002、20131003；In Fig. 6: 1 is 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-reference substance, 2 for lacking Rhizoma Curcumae negative control, and 3,4,5 respectively treat uterus The Chinese medicine granules test sample 20131001,20131002,20131003 of endometrium ectopia；In Fig. 7: 1 is Poria control medicinal material, 2 for lack Poria negative control, 3,4,5 be respectively treatment endometriosis Chinese medicine granules test sample 20131001, 20131002、20131003。

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is further introduced.

Embodiment 1 treats the discriminating of Ramulus Cinnamomi in the Chinese medicine preparation of endometriosis:

The discrimination method of Ramulus Cinnamomi is with cinnamic aldehyde reference substance for comparison, with petroleum ether-acetic acid second that boiling range is 60～90 DEG C Ester=17:3 is the thin layer chromatography of developing solvent, particularly as follows: take this preparation or its content 3g, adds ethanol 10mL, close plug, soaks 20 minutes, shaking constantly, filter, filtrate is as need testing solution；Separately take cinnamic aldehyde reference substance, add ethanol and make every 1mL containing 1 μ L Solution, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw test sample Solution 10～15 μ L, reference substance solution 2 μ L, put on same silica gel g thin-layer plate, with petroleum ether-vinegar that boiling range is 60～90 DEG C Acetoacetic ester=17:3 is developing solvent, launches, and takes out, dries, and spray is with dinitrophenylhydrazine ethanol test solution；In test sample chromatograph, with On the corresponding position of reference substance chromatograph, the speckle of aobvious same color.

Embodiment 2 treats the discriminating of the Radix Aucklandiae in the Chinese medicine preparation of endometriosis:

The discrimination method of the Radix Aucklandiae be with dehydrocostuslactone reference substance, costunolide reference substance for comparison, with hexamethylene- The thin layer chromatography that upper solution is developing solvent of Ethyl formate-formic acid=15:5:1, particularly as follows: take this preparation or its content 4g, adds methanol 10mL, supersound process 30 minutes, filters, take filtrate as need testing solution；Separately take dehydrocostuslactone comparison Product, costunolide reference substance, add methanol and be respectively prepared every 1mL solution containing 0.5mg, as reference substance solution；According to middle traditional Chinese medicines Allusion quotation one annex VI B thin layer chromatography test of version in 2010, draws each 5 μ L of above-mentioned three kinds of solution, puts in same silica gel G thin respectively On laminate, with the upper solution of cyclohexane-carboxylic acid ethyl ester-formic acid=15:5:1 as developing solvent, launch, take out, dry, spray with 1% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, on position corresponding with reference substance chromatograph, The speckle of aobvious same color.

Embodiment 3 treats the discriminating of Fructus Corni in the Chinese medicine preparation of endometriosis:

The discrimination method of Fructus Corni is with ursolic acid reference substance for comparison, with toluene-ethyl acetate-formic acid=20:4:0.5 For the thin layer chromatography of developing solvent, particularly as follows: take this preparation or its content 3g, add ethyl acetate 10mL, supersound process 15 points Clock, filters, and filtrate is evaporated, and residue adds dehydrated alcohol 2mL makes dissolving, as need testing solution；Separately take ursolic acid reference substance, add nothing Water-ethanol makes every 1mL solution containing 1mg, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer color of version in 2010 Spectrometry is tested, and draws each 5 μ L of above two solution, puts respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid =20:4:0.5 is developing solvent, launches, and takes out, dries, and spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C Clearly；In test sample chromatograph, on position corresponding with reference substance chromatograph, aobvious identical aubergine speckle.

Embodiment 4 treats the discriminating of Rhizoma Curcumae in the Chinese medicine preparation of endometriosis:

The discrimination method of Rhizoma Curcumae is with 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-reference substance for comparison, with petroleum ether-acetone-second that boiling range is 30～60 DEG C Acetoacetic ester=94:5:1 is the thin layer chromatography of developing solvent, particularly as follows: take this preparation or its content 3g, puts tool plug centrifuge tube In, adding the petroleum ether 10mL that boiling range is 30～60 DEG C, supersound process 20 minutes, filter, filtrate volatilizes, and residue adds dehydrated alcohol 1mL makes dissolving, as need testing solution；Separately take 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-reference substance, add dehydrated alcohol and make every 1mL solution containing 0.4mg, make For reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw above two solution each 10 μ L, puts respectively on same silica gel g thin-layer plate, with petroleum ether-acetone and ethyl acetate=94:5:1 that boiling range is 30～60 DEG C is Developing solvent, launches, and takes out, dries, and spray, with 1% vanillin-sulfuric acid solution, is heated to spot development at 105 DEG C clear；Test sample In chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.

Embodiment 5 treats the discriminating of Poria in the Chinese medicine preparation of endometriosis:

The discrimination method of Poria is with Poria control medicinal material for comparison, with toluene-ethyl acetate-formic acid=20:5:0.5 is The thin layer chromatography of developing solvent, particularly as follows: take this preparation or its content 4g, add diethyl ether 50mL, supersound process 10 minutes, filter Crossing, filtrate is evaporated, and residue adds methanol 1mL makes dissolving, as need testing solution；Separately taking Poria control medicinal material lg, it is right to be made in the same way of According to medical material solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw each 2 μ L of above two solution, Put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20:5:0.5 as developing solvent, launch, take out, dry in the air Dry, spray, with 2% vanillin-sulfuric acid solution-ethanol=4:1 mixed solution, is heated to spot development at 105 DEG C clear；Test sample color In spectrum, on position corresponding with reference substance chromatograph, the principal spot of aobvious same color.

Embodiment 6 treats the discriminating of Cortex Moutan in the Chinese medicine preparation of endometriosis:

The discrimination method of Cortex Moutan is with paeonol reference substance for comparison, with cyclohexane-ethyl acetate-glacial acetic acid=4:1: The thin layer chromatography that 0.1 is developing solvent, particularly as follows: take this preparation or its content 4g, add diethyl ether l0mL, close plug, shakes 10 points Clock, filters, and filtrate volatilizes, and residue adds acetone 2mL makes dissolving, as need testing solution；Separately take paeonol reference substance, add acetone system Become every 1mL solution containing 2mg as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, Draw each 10 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate-glacial acetic acid=4: 1:0.1 is developing solvent, launches, and takes out, dries, and spray, with 2% vanillin-sulfuric acid ethanol solution (1 → l0), is heated to speckle at 105 DEG C Point colour developing is clear；In test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.

Embodiment 7 treats the discriminating of Radix Paeoniae Rubra in the Chinese medicine preparation of endometriosis:

The discrimination method of Radix Paeoniae Rubra is with peoniflorin reference substance for comparison, with chloroform-ethyl acetate-methyl alcohol-formic acid=40:5: 10:0.2 is the thin layer chromatography of developing solvent, particularly as follows: take this preparation or its content 3g, adds ethanol 10mL, shakes 5 minutes, Filtering, filtrate is evaporated, and residue adds ethanol 2mL makes dissolving, as need testing solution；Separately take peoniflorin reference substance, add ethanol and make Every 1mL solution containing 2mg, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, Draw each 4 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methyl alcohol-formic acid= 40:5:10:0.2 is developing solvent, launches, and takes out, dries, and spray, with 5% vanillin-sulfuric acid solution, is heated to spot development clear； In test sample chromatograph, on position corresponding with reference substance chromatograph, aobvious identical bluish violet speckle.

Embodiment 8 treats the assay of Radix Paeoniae Rubra in the Chinese medicine preparation of endometriosis:

Radix Paeoniae Rubra content assaying method is with peoniflorin reference substance for comparison, with methanol-0.05mol/L potassium dihydrogen phosphate =40:65 is the high performance liquid chromatography of flowing phase, and concrete content assaying method is:

Radix Paeoniae Rubra is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol- 0.05mol/L potassium dihydrogen phosphate=40:65 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 230nm, number of theoretical plate should be not less than 3000 based on peoniflorin peak；

It is appropriate that the preparation of reference substance solution takes peoniflorin reference substance, accurately weighed, adds methanol and makes every 1mL containing peoniflorin The solution of 30 μ g, to obtain final product；

The preparation of need testing solution takes this preparation or its content 1g, accurately weighed, to 50mL conical flask, adds first Alcohol extraction solvent about 25mL, supersound extraction 10min, take out, filter, take subsequent filtrate, to obtain final product；

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

Embodiment 9 treats the assay of Herba Polygoni cymosi in the Chinese medicine preparation of endometriosis:

Herba Polygoni cymosi content assaying method is with Quercetin reference substance for comparison, with methanol-0.4% phosphoric acid solution=50:50 For the high performance liquid chromatography of the phase that flows, concrete content assaying method is:

Herba Polygoni cymosi is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol-0.4% Phosphoric acid solution=50:50 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 360nm；Number of theoretical plate presses Cortex querci dentatae Element peak meter should be not less than 3000；

It is appropriate that the preparation of reference substance solution takes Quercetin reference substance, accurately weighed, adds methanol and makes every 1mL containing Quercetin The solution of 30 μ g, to obtain final product；

The preparation of need testing solution takes this preparation or its content 2g, accurately weighed, to round-bottomed flask, and addition hydrochloric acid: The mixed solution 50mL of methanol=1:4, weighed weight, put 90 DEG C of water-bath reflux, extract, 1 hour, take out, cooling, weighed weight, Supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；

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

The detection method of the Chinese medicine preparation for the treatment of endometriosis of the present invention includes differentiating and assay item Mesh, wherein differentiates at least to include any one in embodiment 1～embodiment 5, assay at least includes embodiment 8～embodiment Any one in 9.

The detection method of the Chinese medicine granules that embodiment 10 treats endometriosis includes:

Character: this preparation is yellowish-brown granule；Feeble QI, mildly bitter flavor；

Differentiate:

(1) taking this preparation 3g, add ethanol 10mL, close plug, soak 20 minutes, shake constantly, filter, filtrate is as test sample Solution；Separately take cinnamic aldehyde reference substance, add ethanol and make every 1mL solution containing 1 μ L, as reference substance solution；According to Chinese Pharmacopoeia One annex VI B thin layer chromatography test of version in 2010, draws need testing solution 10～15 μ L, reference substance solution 2 μ L, puts in same On one silica gel g thin-layer plate, with petroleum ether-ethyl acetate=17:3 that boiling range is 60～90 DEG C as developing solvent, launch, take out, dry in the air Dry, spray is with dinitrophenylhydrazine ethanol test solution；In test sample chromatograph, on position corresponding with reference substance chromatograph, aobvious same color Speckle；

(2) take this preparation 4g, add methanol 10mL, supersound process 30 minutes, filter, take filtrate as need testing solution；Separately Take dehydrocostuslactone reference substance, costunolide reference substance, add methanol and be respectively prepared every 1mL solution containing 0.5mg, as right According to product solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw each 5 μ L of above-mentioned three kinds of solution, point Other point is on same silica gel g thin-layer plate, with the upper solution of cyclohexane-carboxylic acid ethyl ester-formic acid=15:5:1 as developing solvent, and exhibition Opening, take out, dry, spray, with 1% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, with reference substance On the corresponding position of chromatograph, the speckle of aobvious same color；

(3) taking this preparation 3g, add ethyl acetate 10mL, supersound process 15 minutes, filter, filtrate is evaporated, and residue adds anhydrous Ethanol 2mL makes dissolving, as need testing solution；Separately take ursolic acid reference substance, add dehydrated alcohol and make every 1mL solution containing 1mg, As reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw above two solution each 5 μ L, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20:4:0.5 as developing solvent, launch, take Going out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear；In test sample chromatograph, with compare On the corresponding position of product chromatograph, aobvious identical aubergine speckle；

(4) take this preparation 3g, put in tool plug centrifuge tube, add the petroleum ether 10mL that boiling range is 30～60 DEG C, supersound process 20 Minute, filtering, filtrate volatilizes, and residue adds dehydrated alcohol 1mL makes dissolving, as need testing solution；Separately take 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-reference substance, add Dehydrated alcohol makes every 1mL solution containing 0.4mg, as reference substance solution；Thin according to Chinese Pharmacopoeia one annex VI B of version in 2010 Layer chromatography is tested, and draws each 10 μ L of above two solution, puts respectively on same silica gel g thin-layer plate, be 30～60 with boiling range DEG C petroleum ether-acetone and ethyl acetate=94:5:1 be developing solvent, launch, take out, dry, spray molten with 1% vanillin-sulfuric acid Liquid, is heated to spot development at 105 DEG C clear；In test sample chromatograph, on position corresponding with reference substance chromatograph, aobvious identical face The speckle of color；

(5) taking this preparation 4g, add diethyl ether 50mL, supersound process 10 minutes, filters, and filtrate is evaporated, and residue adds methanol 1mL to be made Dissolve, as need testing solution；Separately take Poria control medicinal material lg, be made in the same way of control medicinal material solution；According to Chinese Pharmacopoeia 2010 One annex VI B thin layer chromatography test of version, draws each 2 μ L of above two solution, puts respectively on same silica gel g thin-layer plate, With toluene-ethyl acetate-formic acid=20:5:0.5 as developing solvent, launch, take out, dry, spray with 2% vanillin-sulfuric acid solution- Ethanol=4:1 mixed solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, corresponding with reference substance chromatograph On position, the principal spot of aobvious same color；

(6) taking this preparation 4g, add diethyl ether l0mL, close plug, shakes 10 minutes, filters, and filtrate volatilizes, and residue adds acetone 2mL Make dissolving, as need testing solution；Separately taking paeonol reference substance, adding acetone, to make every 1mL molten as reference substance containing the solution of 2mg Liquid；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw each 10 μ L of above two solution, point respectively On same silica gel g thin-layer plate, with cyclohexane-ethyl acetate-glacial acetic acid=4:1:0.1 as developing solvent, launch, take out, dry, Spray, with 2% vanillin-sulfuric acid ethanol solution (1 → l0), is heated to spot development at 105 DEG C clear；In test sample chromatograph, with On the corresponding position of reference substance chromatograph, the speckle of aobvious same color；

(7) taking this preparation 3g, add ethanol 10mL, shake 5 minutes, filter, filtrate is evaporated, and residue adds ethanol 2mL makes dissolving, As need testing solution；Separately take peoniflorin reference substance, add ethanol and make every 1mL solution containing 2mg, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draws each 4 μ L of above two solution, puts respectively in same On silica gel g thin-layer plate, with chloroform-ethyl acetate-methyl alcohol-formic acid=40:5:10:0.2 as developing solvent, launch, take out, dry, Spray, with 5% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, in position corresponding with reference substance chromatograph Put, aobvious identical bluish violet speckle；

Check: every regulation relevant under Chinese Pharmacopoeia one annex I C granule item of version in 2010 should be met；

Assay: (1) Radix Paeoniae Rubra is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol- 0.05mol/L potassium dihydrogen phosphate=40:65 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 230nm, number of theoretical plate should be not less than 3000 based on peoniflorin peak；

It is appropriate that the preparation of reference substance solution takes peoniflorin reference substance, accurately weighed, adds methanol and makes every 1mL containing peoniflorin The solution of 30 μ g, to obtain final product；

The preparation of need testing solution takes this preparation 1g, accurately weighed, to 50mL conical flask, adds methanol extraction solvent About 25mL, supersound extraction 10min, take out, filter, take subsequent filtrate, to obtain final product；

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

This granule every bag in terms of peoniflorin, must not be less than 11.8mg containing Radix Paeoniae Rubra.

(2) Herba Polygoni cymosi is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol-0.4% Phosphoric acid solution=50:50 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 360nm；Number of theoretical plate presses Cortex querci dentatae Element peak meter should be not less than 3000；

It is appropriate that the preparation of reference substance solution takes Quercetin reference substance, accurately weighed, adds methanol and makes every 1mL containing Quercetin The solution of 30 μ g, to obtain final product；

The preparation of need testing solution takes this preparation 2g, accurately weighed, to round-bottomed flask, and addition hydrochloric acid: methanol=1:4 Mixed solution 50mL, weighed weight, put 90 DEG C of water-bath reflux, extract, 1 hour, take out, cooling, weighed weight, supply with methanol The weight of less loss, shakes up, and filters, takes subsequent filtrate, to obtain final product；

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

This granule every bag in terms of Quercetin, must not be less than 0.5mg containing Herba Polygoni cymosi.

Claims (9)

1. the detection method of the Chinese medicine preparation treating endometriosis, it is characterised in that: described detection method includes Differentiate and assay project；Discriminating is the mirror of the thin layer chromatography to Radix Paeoniae Rubra, Cortex Moutan, Ramulus Cinnamomi, the Radix Aucklandiae, Fructus Corni, Rhizoma Curcumae, Poria Not；Assay is with Radix Paeoniae Rubra, the content of Herba Polygoni cymosi in Preparations by HPLC.

The detection method of the Chinese medicine preparation for the treatment of endometriosis the most according to claim 1, it is characterised in that: osmanthus The discrimination method of branch is with cinnamic aldehyde reference substance for comparison, with petroleum ether-ethyl acetate=17:3 that boiling range is 60～90 DEG C is The thin layer chromatography of developing solvent；The discrimination method of the Radix Aucklandiae is to be right with dehydrocostuslactone reference substance, costunolide reference substance According to, the thin layer chromatography with the upper solution of cyclohexane-carboxylic acid ethyl ester-formic acid=15:5:1 as developing solvent；The discriminating of Fructus Corni Method is with ursolic acid reference substance for comparison, with toluene-ethyl acetate-formic acid=20:4:0.5 thin layer chromatography as developing solvent Method；The discrimination method of Rhizoma Curcumae is with 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-reference substance for comparison, with petroleum ether-acetone-acetic acid second that boiling range is 30～60 DEG C Ester=94:5:1 is the thin layer chromatography of developing solvent；The discrimination method of Poria is with Poria control medicinal material for comparison, with toluene-second Acetoacetic ester-formic acid=20:5:0.5 is the thin layer chromatography of developing solvent.

The detection method of the Chinese medicine preparation for the treatment of endometriosis the most according to claim 2, it is characterised in that: tool Body discrimination method includes at least one in following project:

(1) Ramulus Cinnamomi takes this preparation or its content 3g, adds ethanol 10mL, close plug, soaks 20 minutes, shake constantly, filters, filter Liquid is as need testing solution；Separately take cinnamic aldehyde reference substance, add ethanol and make every 1mL solution containing 1 μ L, as reference substance solution； According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw need testing solution 10～15 μ L, reference substance solution 2 μ L, put on same silica gel g thin-layer plate, with petroleum ether-ethyl acetate=17:3 that boiling range is 60～90 DEG C as developing solvent, and exhibition Opening, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution；In test sample chromatograph, on position corresponding with reference substance chromatograph, The speckle of aobvious same color；

(2) Radix Aucklandiae takes this preparation or its content 4g, adds methanol 10mL, supersound process 30 minutes, filters, take filtrate as confession Test sample solution；Separately take dehydrocostuslactone to add methanol according to product, costunolide reference substance and be respectively prepared molten containing 0.5mg of every 1mL Liquid, as reference substance solution；According to one the annex VI B thin layer chromatography test of Chinese Pharmacopoeia version in 2010, draw above-mentioned three kinds molten The each 5 μ L of liquid, put respectively on same silica gel g thin-layer plate, with the upper solution of cyclohexane-carboxylic acid ethyl ester-formic acid=15:5:1 are Developing solvent, launches, and takes out, dries, and spray, with 1% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, On position corresponding with reference substance chromatograph, the speckle of aobvious same color；

(3) Fructus Corni takes this preparation or its content 3g, adds ethyl acetate 10mL, supersound process 15 minutes, filters, and filtrate is steamed Dry, residue adds dehydrated alcohol 2mL makes dissolving, as need testing solution；Separately take ursolic acid reference substance, add dehydrated alcohol and make often The 1mL solution containing 1mg, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, inhale Take each 5 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20:4:0.5 For developing solvent, launching, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear；Test sample In chromatograph, on position corresponding with reference substance chromatograph, aobvious identical aubergine speckle；

(4) Rhizoma Curcumae takes this preparation or its content 3g, puts in tool plug centrifuge tube, adds the petroleum ether 10mL that boiling range is 30～60 DEG C, Supersound process 20 minutes, filters, and filtrate volatilizes, and residue adds dehydrated alcohol 1mL makes dissolving, as need testing solution；Separately take lucky horse Ketone reference substance, adds dehydrated alcohol and makes every 1mL solution containing 0.4mg, as reference substance solution；According to Chinese Pharmacopoeia version one in 2010 Portion's annex VI B thin layer chromatography is tested, and draws each 10 μ L of above two solution, puts respectively on same silica gel g thin-layer plate, with boiling Journey be the petroleum ether-acetone and ethyl acetate=94:5:1 of 30～60 DEG C be developing solvent, launch, take out, dry, spray with 1% Rhizoma et radix valerianae Aldehyde sulfuric acid solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, on position corresponding with reference substance chromatograph, The speckle of aobvious same color；

(5) Poria takes this preparation or its content 4g, and add diethyl ether 50mL, supersound process 10 minutes, filters, and filtrate is evaporated, residue Add methanol 1mL and make dissolving, as need testing solution；Separately take Poria control medicinal material lg, be made in the same way of control medicinal material solution；According to China Pharmacopeia one annex VI B thin layer chromatography test of version in 2010, draws each 2 μ L of above two solution, puts respectively in same silica gel G On lamellae, with toluene-ethyl acetate-formic acid=20:5:0.5 as developing solvent, launching, take out, dry, spray is with 2% vanillin Sulfuric acid solution-ethanol=4:1 mixed solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, with reference substance On the corresponding position of chromatograph, the principal spot of aobvious same color.

4., according to the detection method of the Chinese medicine preparation treating endometriosis described in claim 1,2 or 3, its feature exists In: the discrimination method of Radix Paeoniae Rubra is with peoniflorin reference substance for comparison, with chloroform-ethyl acetate-methyl alcohol-formic acid=40:5:10: The thin layer chromatography that 0.2 is developing solvent；The discrimination method of Cortex Moutan is with paeonol reference substance for comparison, with hexamethylene-acetic acid Ethyl ester-glacial acetic acid=4:1:0.1 is the thin layer chromatography of developing solvent.

The detection method of the Chinese medicine preparation for the treatment of endometriosis the most according to claim 4, it is characterised in that: male The discrimination method of Cortex Moutan is particularly as follows: take this preparation or its content 4g, and add diethyl ether l0mL, close plug, shakes 10 minutes, filters, filter Liquid volatilizes, and residue adds acetone 2mL makes dissolving, as need testing solution；Separately take paeonol reference substance, add acetone and make every 1mL and contain The solution of 2mg is as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw above-mentioned Two kinds of each 10 μ L of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate-glacial acetic acid=4:1:0.1 are Developing solvent, launches, and takes out, dries, and spray, with 2% vanillin-sulfuric acid ethanol solution, is heated to spot development at 105 DEG C clear；Supply In test product chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.

The detection method of the Chinese medicine preparation for the treatment of endometriosis the most according to claim 4, it is characterised in that: red The discrimination method of Chinese herbaceous peony, particularly as follows: take this preparation or its content 3g, adds ethanol 10mL, shakes 5 minutes, filters, and filtrate is evaporated, residual Slag adds ethanol 2mL makes dissolving, as need testing solution；Separately take peoniflorin reference substance, add ethanol and make every 1mL solution containing 2mg, As reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw above two solution each 4 μ L, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methyl alcohol-formic acid=40:5:10:0.2 as developing solvent, Launching, take out, dry, spray, with 5% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, with compare On the corresponding position of product chromatograph, aobvious identical bluish violet speckle.

The detection method of the Chinese medicine preparation for the treatment of endometriosis the most according to claim 1, it is characterised in that: red The content assaying method of Chinese herbaceous peony is with peoniflorin reference substance for comparison, with methanol-0.05mol/L potassium dihydrogen phosphate=40:65 High performance liquid chromatography for the phase that flows；The content assaying method of Herba Polygoni cymosi be with Quercetin reference substance for comparison, with methanol- 0.4% phosphoric acid solution=50:50 is the high performance liquid chromatography of flowing phase.

The detection method of the Chinese medicine preparation for the treatment of endometriosis the most according to claim 7, it is characterised in that: tool The content assaying method of body includes at least one in following project:

(1) Radix Paeoniae Rubra is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol-0.05mol/L Potassium dihydrogen phosphate=40:65 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 230nm, number of theoretical plate 3000 should be not less than based on peoniflorin peak；

It is appropriate that the preparation of reference substance solution takes peoniflorin reference substance, accurately weighed, adds methanol and makes every 1mL μ g Han peoniflorin 30 Solution, to obtain final product；

The preparation of need testing solution takes this preparation or its content 1g, accurately weighed, to 50mL conical flask, adds methanol and carries Take solvent 25mL, supersound extraction 10min, take out, filter, take subsequent filtrate, to obtain final product；

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

(2) Herba Polygoni cymosi is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol-0.4% phosphoric acid Solution=50:50 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 360nm；Number of theoretical plate presses Quercetin peak Meter should be not less than 3000；

It is appropriate that the preparation of reference substance solution takes Quercetin reference substance, accurately weighed, adds methanol and makes every 1mL μ g Han Quercetin 30 Solution, to obtain final product；

The preparation of need testing solution takes this preparation or its content 2g, accurately weighed, to round-bottomed flask, and addition hydrochloric acid: methanol The mixed solution 50mL of=1:4, weighed weight, put 90 DEG C of water-bath reflux, extract, 1 hour, take out, cooling, weighed weight, use first The weight of less loss supplied by alcohol, shakes up, and filters, takes subsequent filtrate, to obtain final product；

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

9. according to claims 1 to 3, the detection side of the Chinese medicine preparation for the treatment of endometriosis according to any one of 7～8 Method, it is characterised in that: described detection method includes:

Differentiate: (1) takes this preparation or its content 3g, adds ethanol 10mL, close plug, soaks 20 minutes, shake constantly, filter, filter Liquid is as need testing solution；Separately take cinnamic aldehyde reference substance, add ethanol and make every 1mL solution containing 1 μ L, as reference substance solution； According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw need testing solution 10～15 μ L, reference substance solution 2 μ L, put on same silica gel g thin-layer plate, with petroleum ether-ethyl acetate=17:3 that boiling range is 60～90 DEG C as developing solvent, and exhibition Opening, take out, dry, spray is with dinitrophenylhydrazine ethanol test solution；In test sample chromatograph, on position corresponding with reference substance chromatograph, The speckle of aobvious same color；

(2) take this preparation or its content 4g, add methanol 10mL, supersound process 30 minutes, filter, take filtrate molten as test sample Liquid；Separately take dehydrocostuslactone reference substance, costunolide reference substance, add methanol and be respectively prepared every 1mL solution containing 0.5mg, As reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw above-mentioned three kinds of solution each 5 μ L, put respectively on same silica gel g thin-layer plate, with the upper solution of cyclohexane-carboxylic acid ethyl ester-formic acid=15:5:1 for launching Agent, launches, and takes out, dries, and spray, with 1% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, with On the corresponding position of reference substance chromatograph, the speckle of aobvious same color；

(3) taking this preparation or its content 3g, add ethyl acetate 10mL, supersound process 15 minutes, filter, filtrate is evaporated, residue Add dehydrated alcohol 2mL and make dissolving, as need testing solution；Separately take ursolic acid reference substance, add dehydrated alcohol and make every 1mL containing 1mg Solution, as reference substance solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw above-mentioned two Plant each 5 μ L of solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20:4:0.5 as developing solvent, Launching, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear；In test sample chromatograph, On position corresponding with reference substance chromatograph, aobvious identical aubergine speckle；

(4) take this preparation or its content 3g, put in tool plug centrifuge tube, add the petroleum ether 10mL that boiling range is 30～60 DEG C, ultrasonic Processing 20 minutes, filter, filtrate volatilizes, and residue adds dehydrated alcohol 1mL makes dissolving, as need testing solution；Separately take 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-pair According to product, add dehydrated alcohol and make every 1mL solution containing 0.4mg, as reference substance solution；Attached according to Chinese Pharmacopoeia version one in 2010 Record VI B thin layer chromatography test, draw each 10 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with boiling range be Petroleum ether-acetone and ethyl acetate=the 94:5:1 of 30～60 DEG C is developing solvent, launches, and takes out, dries, and spray is with 1% vanillin sulfur Acid solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, on position corresponding with reference substance chromatograph, aobvious phase Speckle with color；

(5) taking this preparation or its content 4g, add diethyl ether 50mL, supersound process 10 minutes, filters, and filtrate is evaporated, and residue adds first Alcohol 1mL makes dissolving, as need testing solution；Separately take Poria control medicinal material lg, be made in the same way of control medicinal material solution；According to Chinese Pharmacopoeia One annex VI B thin layer chromatography test of version in 2010, draws each 2 μ L of above two solution, puts respectively in same silica gel G thin layer On plate, with toluene-ethyl acetate-formic acid=20:5:0.5 as developing solvent, launching, take out, dry, spray is with 2% vanillin-sulfuric acid Solution-ethanol=4:1 mixed solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, with reference substance chromatograph On corresponding position, the principal spot of aobvious same color；

(6) taking this preparation or its content 4g, add diethyl ether l0mL, close plug, shakes 10 minutes, filters, and filtrate volatilizes, and residue adds the third Ketone 2mL makes dissolving, as need testing solution；Separately take paeonol reference substance, add acetone and make every 1mL solution containing 2mg as right According to product solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw each 10 μ L of above two solution, Put respectively on same silica gel g thin-layer plate, with cyclohexane-ethyl acetate-glacial acetic acid=4:1:0.1 as developing solvent, launch, take Going out, dry, spray, with 2% vanillin-sulfuric acid ethanol solution, is heated to spot development at 105 DEG C clear；In test sample chromatograph, with On the corresponding position of reference substance chromatograph, the speckle of aobvious same color；

(7) taking this preparation or its content 3g, add ethanol 10mL, shake 5 minutes, filter, filtrate is evaporated, and residue adds ethanol 2mL Make dissolving, as need testing solution；Separately take peoniflorin reference substance, add ethanol and make every 1mL solution containing 2mg, as reference substance Solution；According to Chinese Pharmacopoeia one annex VI B thin layer chromatography test of version in 2010, draw each 4 μ L of above two solution, point respectively On same silica gel g thin-layer plate, with chloroform-ethyl acetate-methyl alcohol-formic acid=40:5:10:0.2 as developing solvent, launch, take out, Drying, spray, with 5% vanillin-sulfuric acid solution, is heated to spot development clear；In test sample chromatograph, corresponding to reference substance chromatograph Position on, aobvious identical bluish violet speckle；

Assay: (1) Radix Paeoniae Rubra is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol-0.05mol/L Potassium dihydrogen phosphate=40:65 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 230nm, number of theoretical plate 3000 should be not less than based on peoniflorin peak；

It is appropriate that the preparation of reference substance solution takes peoniflorin reference substance, accurately weighed, adds methanol and makes every 1mL μ g Han peoniflorin 30 Solution, to obtain final product；

The preparation of need testing solution takes this preparation or its content 1g, accurately weighed, to 50mL conical flask, adds methanol and carries Take solvent about 25mL, supersound extraction 10min, take out, filter, take subsequent filtrate, to obtain final product；

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

(2) Herba Polygoni cymosi is according to Chinese Pharmacopoeia one annex VI D high effective liquid chromatography for measuring of version in 2010；

Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With methanol-0.4% phosphoric acid Solution=50:50 is flowing phase；Flow velocity is 1mL/min；Column temperature 30 DEG C；Detection wavelength is 360nm；Number of theoretical plate presses Quercetin peak Meter should be not less than 3000；

It is appropriate that the preparation of reference substance solution takes Quercetin reference substance, accurately weighed, adds methanol and makes every 1mL μ g Han Quercetin 30 Solution, to obtain final product；

The preparation of need testing solution takes this preparation or its content 2g, accurately weighed, to round-bottomed flask, and addition hydrochloric acid: methanol The mixed solution 50mL of=1:4, weighed weight, put 90 DEG C of water-bath reflux, extract, 1 hour, take out, cooling, weighed weight, use first The weight of less loss supplied by alcohol, shakes up, and filters, takes subsequent filtrate, to obtain final product；

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