CN102579861A

CN102579861A - Method for detecting quality of An'erning granules

Info

Publication number: CN102579861A
Application number: CN2011100094359A
Authority: CN
Inventors: 张义智; 马振元; 李怀平; 李丽; 孙绪丁
Original assignee: Shandong Arura Pharmaceutical Research & Development Co Ltd
Current assignee: JINHE TIBETAN MEDICINE CO., LTD.
Priority date: 2011-01-17
Filing date: 2011-01-17
Publication date: 2012-07-18
Anticipated expiration: 2031-01-17
Also published as: CN102579861B

Abstract

The invention discloses a method for detecting the quality of An'erning granules. The method comprises a method for identifying cholic acid and bergenin and a method for determining content of glycyrrhizic acid, the bergenin and bilirubin. Experimental study proves that spots with different colors are displayed at a position, which corresponds to a color spectrum of a reference substance, in the color spectrum of a sample by the identification method in the quality detection method; and negative control is not interfered. Results show that the content determination method is used for determining the content of the glycyrrhizic acid and the bergenin in the An'erning granules, has a good linear relation, and high repeatability and stability, and can control the quality of An' erning granules accurately and strictly; and moreover, the accuracy of recovery tests is high.

Description

The peaceful particulate quality determining method of a kind of peace youngster

Technical field

The present invention relates to a kind of quality determining method of pharmaceutical composition, particularly the particulate quality determining method of An Erning.

Background technology

The peaceful granule of peace youngster is recorded in the national drug standards, standard numbering: WS-10640 (ZD-0640)-2002.Has clearing away heat and expelling wind, the effect of preventing phlegm from forming and stopping coughing.Be used for infantile anemopyretic cold, bring up phlegm when one coughs, the heating pharyngalgia, upper respiratory tract infection is seen above-mentioned patient.

Infant upper respiratory tract infection, flu, cough with asthma are commonly encountered diseases, frequently-occurring diseases clinically, and Therapeutic Method is a lot of, but effect differs.The peaceful granule of peace youngster is Tibetan medicine's traditional ancient proved recipe, and through Tibetan medicine's long-term clinical, this medical instrument has the effect of heat-clearing and toxic substances removing dispelling pathogenic wind and eliminating phlegm, is used for children's's Common Cold, pneumonia, bronchitis and diseases such as cough that causes and heating thereof.

Pacify the peaceful granule of youngster owing to respond well, and taken all factors into consideration children's's physiology, pathological characteristic, evident in efficacy through clinical verification, have no side effect, enjoy the patient to favor clinically.

Yet the peaceful particulate quality inspection standard of existing peace youngster comprises the discriminating of cholic acid, these two parts of bilirubinic assay, and this quality determining method can not guarantee to pacify the peaceful particulate quality of youngster, for hidden danger has been buried in clinical use.Need research one cover badly about peaceful particulate more accurate, the tight quality determining method of peace youngster.

Summary of the invention

The object of the invention is to disclose the peaceful particulate quality determining method of a kind of peace youngster.

The present invention seeks to realize through following scheme.

The present invention pacifies the peaceful particulate quality determining method of youngster and comprises in following discriminating and/or the content assaying method one or more:

Differentiate:

A. get the peaceful granule 1-3g of peace youngster, porphyrize adds acetone 5-15ml, and supersound process 10-30 minute, filter, filtrating is concentrated into 1ml, as need testing solution; Other gets cholic acid, Hyodeoxycholic Acid reference substance, adds acetone and processes the mixed solution that every 1ml contains 0.1-0.3mg, as reference substance solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With E-C-glacial acetic acid (1-3: 1-3: 0.5-1.5) be developing solvent, launch, take out; Dry; Spray, is put under the ultra-violet lamp (365nm) and is inspected 100 ℃-110 ℃ heating several minutes with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

B. after pacifying the peaceful granule porphyrize of youngster, get powder 5-15g, add methanol 15-25ml, supersound extraction 20-40min; Take out, put coldly, filter; After getting the subsequent filtrate evaporate to dryness, residue water 10-30ml all is transferred to separatory funnel, and aqueous solution is with ethyl acetate extraction 3-5 time; Each 10-30ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Other gets the Bergeninum reference substance, adds dissolve with methanol and be diluted to every 1ml methanol solution to contain the 0.1-0.3mg reference substance solution, shakes up, and compares article solution; According to the thin layer chromatography test, draw each 10 μ l of above-mentioned solution, put respectively on same silica GF254 lamellae, with chloroform-ethyl acetate-methanol (3-7: 2-6: 1-4) be developing solvent, launch that taking-up is dried, and puts under the ultra-violet lamp 254nm and inspects; Be in the test sample chromatograph on the corresponding position of reference substance, show the speckle of same color.

Assay

A. bilirubin is measured according to high-efficient liquid phase technique (an appendix VI of Chinese Pharmacopoeia version in 2010 D);

Chromatographic condition and system suitability test: with its silane group silica gel of octadecane is filler; With methanol-oxolane-0.5% acetum (70-90: 5-15: 5-15) be mobile phase; The detection wavelength is 450nm; Number of theoretical plate calculates by the bilirubin peak should be not less than 2000;

The preparation of reference substance solution: precision takes by weighing the bilirubin reference substance, and chlorination is copied into the solution that every 1ml contains 5-15 μ g, promptly gets;

The preparation of need testing solution: behind the peaceful granule porphyrize of peace youngster, get powder 3-7g, the accurate title, decide, and puts in the conical flask of tool plug, the accurate dichloromethane 20-30ml that adds; Close plug is claimed to decide weight, and ultrasonic 20-40min is put cold; Supply the weight that subtracts mistake with dichloromethane, shake up, filter, promptly get;

Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly get;

The every gram of the peace peaceful granule of youngster contains bilirubin (C ₃₃H ₃₆N ₄O ₆) meter, must not be less than 0.04mg;

B. glycyrrhizic acid is measured according to high-efficient liquid phase technique (an appendix VI of Chinese Pharmacopoeia version in 2010 D);

Chromatographic condition and system suitability test: with its silane group silica gel of octadecane is filler; With acetonitrile-0.5% phosphoric acid solution (30-40: 60-70) be mobile phase; The detection wavelength is 250nm; Number of theoretical plate calculates by Radix Glycyrrhizae acid amide peak should be not less than 5000;

The preparation of reference substance solution: precision takes by weighing the ammonium glycyrrhizinate reference substance, adds methanol and processes the solution that every 1ml contains 0.1-0.3mg, promptly gets (glycyrrhizic acid weight=ammonium glycyrrhizinate/1.0207);

The preparation of need testing solution: behind the peaceful granule porphyrize of peace youngster, get powder 5-8g, the accurate title, decide, and puts in the conical flask of tool plug, the accurate methanol 20-30ml that adds; Close plug is claimed to decide weight, and ultrasonic 20-40min is put cold; Supply the weight that subtracts mistake with methanol, shake up, filter, promptly get;

The every gram of the peace peaceful granule of youngster contains glycyrrhizic acid (C ₄₂H ₆₁O ₁₆NH ₄) meter, must not be less than 0.33mg;

C. Bergeninum is measured according to high-efficient liquid phase technique (an appendix VI of Chinese Pharmacopoeia version in 2010 D);

Chromatographic condition and system suitability test: with its silane group silica gel of octadecane is filler; With methanol-water (10-30: 70-90) be mobile phase; The detection wavelength is 275nm; Number of theoretical plate calculates by the Bergeninum peak should be not less than 2000;

The preparation of reference substance solution: precision takes by weighing the Bergeninum reference substance, adds methanol and processes the solution that every 1ml contains 40-60 μ g, promptly gets;

The preparation of need testing solution: behind the peaceful granule porphyrize of peace youngster, get powder 2-4g, the accurate title, decide, and puts in the conical flask of tool plug, the accurate methanol 40-60ml that adds; Close plug is claimed to decide weight, and ultrasonic 20-40min is put cold; Supply the weight that subtracts mistake with methanol, shake up, filter, promptly get;

The every gram of the peace peaceful granule of youngster contains Rhizoma Seu Herba Bergeniae element (C ₁₄H ₁₆O ₉) meter, must not be less than 0.67mg.

The present invention pacifies the peaceful particulate quality determining method of youngster and preferably includes in following discriminating and/or the content assaying method one or more:

Differentiate:

A. get the peaceful granule 2g of peace youngster, porphyrize adds acetone 10ml, and supersound process 20 minutes filters, and filtrating is concentrated into 1ml, as need testing solution; Other gets cholic acid, Hyodeoxycholic Acid reference substance, adds acetone and processes the mixed solution that every 1ml contains 0.2mg, as reference substance solution; According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With E-C-glacial acetic acid (2: 2: 1) is developing solvent, launches, and takes out; Dry; Spray, is put under the ultra-violet lamp (365nm) and is inspected 105 ℃ of heating several minutes with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

B. after pacifying the peaceful granule porphyrize of youngster, get powder 10g, add methanol 20ml, supersound extraction 30min takes out; Put coldly, filter, get the subsequent filtrate evaporate to dryness after, residue water 20ml all is transferred to separatory funnel; Aqueous solution is with ethyl acetate extraction 4 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Other gets the Bergeninum reference substance, adds dissolve with methanol and be diluted to every 1ml methanol solution to contain the 0.2mg reference substance solution, shakes up, and compares article solution; According to the thin layer chromatography test, draw each 10 μ l of above-mentioned solution, put respectively on same silica GF254 lamellae, be developing solvent with chloroform-ethyl acetate-methanol (5: 4: 2.5), launch, taking-up is dried, and puts under the ultra-violet lamp 254nm and inspects; Be in the test sample chromatograph on the corresponding position of reference substance, show the speckle of same color.

Assay:

Chromatographic condition and system suitability test: with its silane group silica gel of octadecane is filler; With methanol-oxolane-0.5% acetum (80: 10: 10) is mobile phase; The detection wavelength is 450nm; Number of theoretical plate calculates by the bilirubin peak should be not less than 2000;

The preparation of reference substance solution: precision takes by weighing the bilirubin reference substance, and chlorination is copied into the solution that every 1ml contains 10 μ g, promptly gets;

The preparation of need testing solution: behind the peaceful granule porphyrize of peace youngster, get powder 5g, the accurate title, decide, and puts in the conical flask of tool plug, the accurate dichloromethane 25ml that adds; Close plug is claimed to decide weight, and ultrasonic 30min is put cold; Supply the weight that subtracts mistake with dichloromethane, shake up, filter, promptly get;

The every gram of the peace peaceful granule of youngster contains bilirubin (C ₃₃H ₃₆N ₄O ₆) meter, must not be less than 0.04mg.

Chromatographic condition and system suitability test: with its silane group silica gel of octadecane is filler; With acetonitrile-0.5% phosphoric acid solution (37: 63) is mobile phase; The detection wavelength is 250nm; Number of theoretical plate calculates by Radix Glycyrrhizae acid amide peak should be not less than 5000;

The preparation of reference substance solution: precision takes by weighing the ammonium glycyrrhizinate reference substance, adds methanol and processes the solution that every 1ml contains 0.2mg, promptly gets (glycyrrhizic acid weight=ammonium glycyrrhizinate/1.0207);

The preparation of need testing solution: behind the peace youngster peaceful granule porphyrize, get powder 6.5g, accurately claim surely, put in the conical flask of tool plug, the accurate methanol 25ml that adds, close plug is claimed decide weight, and ultrasonic 30min is put coldly, supplies the weight that subtracts mistake with methanol, shakes up, and filtration promptly gets;

The every gram of the peace peaceful granule of youngster contains glycyrrhizic acid (C ₄₂H ₆₁O ₁₆NH ₄) meter, must not be less than 0.33mg.

Chromatographic condition and system suitability test: with its silane group silica gel of octadecane is filler; With methanol-water (20: 80) is mobile phase; The detection wavelength is 275nm; Number of theoretical plate calculates by the Bergeninum peak should be not less than 2000;

The preparation of reference substance solution: precision takes by weighing the Bergeninum reference substance, adds methanol and processes the solution that every 1ml contains 50 μ g, promptly gets;

The preparation of need testing solution: behind the peace youngster peaceful granule porphyrize, get powder 3g, accurately claim surely, put in the conical flask of tool plug, the accurate methanol 50ml that adds, close plug is claimed decide weight, and ultrasonic 30min is put coldly, supplies the weight that subtracts mistake with methanol, shakes up, and filtration promptly gets;

Wherein, the peaceful particulate crude drug of above-mentioned peace youngster consists of:

Flos Carthami 30-70 weight portion Santalum album 40-70 weight portion

Radix Glycyrrhizae 30-70 weight portion high mountain Radix Cochleariae officinalis dish 30-70 weight portion

Rhizoma Seu Herba Bergeniae 30-70 weight portion Concretio Silicea Bambusae 40-70 weight portion

Short tube rabbit grass 40-70 weight portion Tangut aconite 40-70 weight portion

Artificial Calculus Bovis 2-8 weight portion sucrose 600-1200 weight portion.

Wherein, the peaceful particulate crude drug composition of above-mentioned peace youngster is preferably:

Flos Carthami 53.3 weight portion Santalum albums 66.7 weight portions

Radix Glycyrrhizae 53.3 weight portion high mountain Radix Cochleariae officinalis dishes 53.3 weight portions

Rhizoma Seu Herba Bergeniae 53.3 weight portion Concretio Silicea Bambusaes 66.7 weight portions

Lagotis brachystachya Maxim 66.7 weight portion Tangut aconite 66.7 weight portions

Artificial Calculus Bovis's 5.3 weight portion sucrose 930 weight portions.

Wherein, the peaceful granule of above-mentioned peace youngster is prepared from following method:

Get the water that Flos Carthami, Santalum album add the 3-7 times of weight and extracted 2-4 hour, collect volatile oil, filter with steam distillation; Medicinal residues and Radix Glycyrrhizae, high mountain Radix Cochleariae officinalis dish, Rhizoma Seu Herba Bergeniae, Concretio Silicea Bambusae, short tube rabbit grass and Tangut aconite merge, add the 6-12 times of weight water, decoct 1-4 time; Time is 1-4 hour, filters merging filtrate; Be concentrated into the clear paste of ρ=1.02～1.04 (50 ℃ of mensuration of temperature), carried out in clear paste to the cold-storage jar cold preservation 12-36 hour, it is centrifugal with the high speed tube centrifuge to get supernatant; Centrifugal liquid continues to be concentrated into thick paste ρ=1.07～1.09 (50 ℃ of mensuration of temperature), and spray drying promptly gets and pacifies the peaceful extract of youngster;

Get beta-schardinger dextrin-5-20 weight portion; Be dissolved in 40 ℃ of water of 100 parts by volume; Ultrasonic 1-3 hour, under powerful magnetic agitation, add the volatile oil ethanolic solution of 1-3 parts by volume, contain the volatile oil of 0.3-0.8 parts by volume in wherein every parts by volume solution; The powerful stirring 1-6 hour; Refrigerate 12-36 hour, filter, 30-50 ℃ of vacuum drying precipitation; Pulverize, promptly get the Benexate Hydrochloride of volatile oil;

Get the peaceful extract of peace youngster, beta-schardinger dextrin-volatile oil clathrate compound and artificial Calculus Bovis, mix and add sucrose, mix homogeneously is done wetting agent with 50% alcoholic solution, and 14 orders are granulated, 50 ℃ of dryings, and 14 order granulate promptly get.

Wherein, the peaceful granule of above-mentioned peace youngster preferably is prepared from following method:

Get the water that Flos Carthami, Santalum album add 5 times of weight and extracted 3 hours, collect volatile oil, filter with steam distillation; Medicinal residues and Radix Glycyrrhizae, high mountain Radix Cochleariae officinalis dish, Rhizoma Seu Herba Bergeniae, Concretio Silicea Bambusae, short tube rabbit grass and Tangut aconite merge, decocte with water 2 times, and the decocting that adds 10 times of weight for the first time boiled 2 hours; The decocting that adds for the second time 8 times of weight boiled 2 hours, filtered merging filtrate; Be concentrated into the clear paste of ρ=1.02～1.04 (50 ℃ of mensuration of temperature), carry out cold preservation 24 hours in clear paste to the cold-storage jar, it is centrifugal with the high speed tube centrifuge to get supernatant; Centrifugal liquid continues to be concentrated into thick paste ρ=1.07～1.09 (50 ℃ of mensuration of temperature), and spray drying promptly gets and pacifies the peaceful extract of youngster;

Get beta-schardinger dextrin-10 weight portions; Be dissolved in 40 ℃ of water of 100 parts by volume; Ultrasonic 2 hours, under powerful magnetic agitation, add the volatile oil ethanolic solution of 2 parts by volume, contain the volatile oil of 0.5 parts by volume in wherein every parts by volume solution; The powerful stirring 2 hours; Refrigerate 24 hours, filter, 40 ℃ of vacuum drying precipitations; Pulverize, promptly get the Benexate Hydrochloride of volatile oil;

The relation of weight portion according to the invention and parts by volume is the relation of g/ml.

The invention discloses a kind ofly about peace youngster peaceful particulate quality determining method, this method comprises the discrimination method of cholic acid and Bergeninum, also comprises glycyrrhizic acid, Bergeninum and bilirubinic content assaying method.Through experimentation, the discrimination method in the quality determining method of the present invention in the test sample chromatograph of sample, with the corresponding position of reference substance chromatograph on, all show the speckle of same color; Negative control is noiseless.With the glycyrrhizic acid in the peaceful granule of content assaying method mensuration peace youngster of the present invention and the content of Bergeninum; The result shows that this method linear relationship is good, and the recovery test accuracy is higher; Have fabulous repeatability and stable, can control the peaceful particulate quality of peace youngster accurate, tightly.

Following experimental example and embodiment are used to further specify but are not limited to the present invention.

The research experiment of 1 pair of various crude drug discrimination method of experimental example

According to being recorded in the national drug standards, standard is numbered: WS-10640 (ZD-0640)-2002, and according to the peaceful granule of the peaceful particulate standard fabrication of peace youngster three batches of peaces youngster, the discrimination method to wherein each kind of crude drug makes an experiment respectively, and experimental basis and result are following:

1, with reference to " a Flos Carthami quality standard of Chinese pharmacopoeia version in 2010 Flos Carthami test, the result shows in three lot sample article and the negative control chromatograph, with the corresponding position of control medicinal material chromatograph on, do not have obvious speckle.So do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

2, with reference to " tlc identification method of compound bergenin pills " to Rhizoma Seu Herba Bergeniae test, the result shows in three lot sample article and the negative control chromatograph, with the corresponding position of reference substance chromatograph on, equal speckles of apparent same color; Negative control has interference.So do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

3, with reference to " content of Bergeninum in the tlc scanning determination lung-clearing anti-inflammatory ball " to Rhizoma Seu Herba Bergeniae test, the result shows in three batches of test sample chromatographs, with the corresponding position of reference substance chromatograph on, all show the speckle of same color; Negative control is noiseless.Pretend to the present invention and pacify the part in the peaceful granular mass detection method of youngster.

4, with reference to " Radix Pecteilis susannae medical material thin layer chromatography differentiate and assay " to the Lagotis brachystachya Maxim test, the result shows in three lot sample article and the negative control chromatograph, with the corresponding position of reference substance chromatograph on, equal speckles of apparent same color; Negative control has interference.So do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

5, with reference to seven flavor Gandanqing capsule quality standard WS-10629 (2D-0629)-2002 pairs of Lagotis brachystachya Maxim tests, the result shows that developing solvent polarity is too little, and each speckle does not almost launch.So do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

6, with reference to " discriminating of ingredient catalpol in the Radix Rehmanniae beverage " to Lagotis brachystachya Maxim test, the result shows in three lot sample article and the negative control chromatograph, with the corresponding position of reference substance chromatograph on, equal speckles of apparent same color; Negative control has interference.So do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

7, with reference to " Concretio Silicea Bambusae quality standard of Chinese pharmacopoeia version in 2010 is tested Concretio Silicea Bambusae, and the result shows that feminine gender has interference.So do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

8, with reference to " Radix Glycyrrhizae quality standard of Chinese pharmacopoeia version in 2010 is tested Radix Glycyrrhizae; The result shows and does not see that ammonium glycyrrhizinate has obvious speckle; The apparent speckle of Radix Glycyrrhizae control medicinal material, test sample also has identical speckle with the negative control corresponding position, so feminine gender has interference.So do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

9, with reference to " thin layer of Radix Glycyrrhizae is differentiated in the compound Radix Glycyrrhizae mixture " Radix Glycyrrhizae is tested, the result shows that test sample also has identical speckle with negative control in the ammonium glycyrrhizinate corresponding position, so feminine gender has interference.So do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

Experimental example 2 HPLC methods are measured the content of glycyrrhizic acid in the peaceful granule of peace youngster

1. instrument, reagent and confession test agent

Instrument: high performance liquid chromatograph: the L-7110 of Hitachi pump, L-7420 UV-detector; Tianjin, island LC-20AT pump, the SPD-20A detector; Tianjin, island AUW220D electronic balance

Reference substance: ammonium glycyrrhizinate reference substance

Sample: the peaceful granule of peace youngster (according to the peaceful particulate standard fabrication of peace youngster that is recorded in the Chinese patent medicine provincial standard rising national drug standards part)

2. the selection of mobile phase

With reference to Radix Glycyrrhizae assay item, intend and select for use acetonitrile-0.5% phosphoric acid solution (37: 63) to be mobile phase, with this understanding, ammonium glycyrrhizinate and other adjacent peak separating degree are bigger, and peak shape is better.

3. detect the selection of wavelength

Extracting liquorice acid ammonium reference substance solution scans in the 200-400nm wave-length coverage, and based on ultraviolet absorpting spectrum, selected 250nm is for detecting wavelength.

4. the preparation of reference substance solution

With reference to the Radix Glycyrrhizae content assaying method, confirm that the method for preparing of reference substance solution is: it is an amount of that precision takes by weighing the ammonium glycyrrhizinate reference substance, adds 70% ethanol and process the solution that every 1ml contains 0.2mg and promptly get.(glycyrrhizic acid weight=ammonium glycyrrhizinate weight/1.0207)

5. the preparation of need testing solution

Get powder 4.0g behind the peace youngster peaceful granule porphyrize, accurately claim surely, put in the conical flask, the accurate 70% ethanol 25ml that adds claims decide weight, supersound process 30min, put cold after, supply the weight that subtracts mistake with 70% ethanol, shake up, filtration promptly gets.

6. system suitability test and negative interferential investigation

Under above-mentioned chromatographic condition; Accurate respectively each the 10 μ l of blank solvent, reference substance solution, need testing solution and negative control solution that draw inject chromatograph of liquid, and the result shows; The separating degree that ammonium glycyrrhizinate is adjacent chromatographic peak in the test sample chromatograph is greater than 1.5, and negative control is noiseless.

7. the investigation of the preparation of standard curve and linear relationship

Precision is measured ammonium glycyrrhizinate reference substance stock solution solution (0.3614mg/ml) 1ml, 2ml, 3ml, 5ml, 8ml, 10ml; Put respectively in the 10ml volumetric flask, 70% ethanol dilution shakes up to scale; Each accurate sample introduction 10 μ l; With peak area (A) reference substance concentration (C) is carried out linear regression, get regression equation: A=7175.9C+43512, correlation coefficient: R=0.9993.The result shows: in 36.14ug/ml～361.40ug/ml scope, the peak area of ammonium glycyrrhizinate (A) is good with concentration (C) linear relationship.

8. precision test

The accurate respectively ammonium glycyrrhizinate reference substance solution 10 μ l that draw inject chromatograph of liquid, each METHOD FOR CONTINUOUS DETERMINATION 6 times, and the record peak area also calculates relative standard deviation, RSD=0.80%.The result shows that instrument precision is good.

9. stability test

After the need testing solution preparation was accomplished, the accurate 10 μ l that draw injected chromatograph of liquid, and the record peak area was whenever measured once at a distance from 2 hours later on, investigated 8 hours, calculated the relative standard deviation of peak area, RSD=0.90%.The result shows: test sample mesobilirubin measurement result in 8 hours is stable.

10. replica test

Get the peaceful granule of peace youngster, replication 6 times, the content of glycyrrhizic acid in the calculation sample.The glycyrrhizic acid average content is as a result: 0.43mg/g, RSD=0.99%.Show that analytical method repeatability is good.

11. recovery test

Precision takes by weighing with 6 parts of the peaceful particulate samples of a collection of peace youngster, and the accurate ammonium glycyrrhizinate reference substance that adds is measured its content, calculate recovery rate, and the result is following.The glycyrrhizic acid average recovery rate is 99.2%, RSD=1.07%.Show that this assay method measurement result is accurate.

Table 1 recovery test result

12. sample determination

Get 3 batches of the peaceful granules of peace youngster, measure and the calculating glycyrrhizic acid content, the result is following.Table 2

The peaceful particulate samples assay of table 2 peace youngster result

Experimental example 3HPLC method is measured the content of Bergeninum in the peaceful granule of peace youngster

1. instrument, reagent and confession test agent

Reference substance: Bergeninum reference substance

2. mobile phase is selected

With reference to " method test under the Bergeninum assay item in Herba Ardisiae Japonicae quality standard of Chinese pharmacopoeia version in 2005, intending and selecting methanol-water (20: 80) is mobile phase.

It is an amount of to get the Bergeninum reference substance, and accurate the title decides, and adds methanol and processes the solution that every 1ml contains 50 μ g, promptly gets.

3, the preparation of Rhizoma Seu Herba Bergeniae blank solution

Prepare the negative control that does not contain Rhizoma Seu Herba Bergeniae by prescription, with the need testing solution manipulation.

Under this chromatographic condition, precision is measured each 10 μ l of above-mentioned solution, injects chromatograph of liquid respectively, and Bergeninum main peak retention time is 13.257min as a result, and number of theoretical plate is 6428.Negative sample is noiseless.

4. detecting wavelength confirms

It is an amount of to get the Bergeninum reference substance; The accurate title, decide; Add the also quantitative dilution of dissolve with methanol and process the solution that contains 20 μ g among every 1ml approximately, in 200～400nm wave-length coverage, carry out UV scanning, the result shows that Bergeninum has absorption maximum in the 273.4nm wavelength; Simultaneously with reference to " content assaying method in Herba Ardisiae Japonicae quality standard of Chinese pharmacopoeia version in 2010 confirms that it is 275nm that these article assay detects wavelength.

5. the preparation of reference substance solution

6. the preparation of need testing solution

Behind the peace youngster peaceful granule porphyrize, get powder 3g, accurately claim surely, put in the conical flask of tool plug, the accurate methanol 50ml that adds, close plug is claimed decide weight, and ultrasonic 30min is put coldly, supplies the weight that subtracts mistake with methanol, shakes up, and filtration promptly gets.

7. system suitability test and negative interferential investigation

Under above-mentioned chromatographic condition; Accurate respectively each the 10 μ l of blank solvent, reference substance solution, need testing solution and negative control solution that draw inject chromatograph of liquid, and the result shows; The separating degree that Bergeninum is adjacent chromatographic peak in the test sample chromatograph is greater than 1.5, and negative control is noiseless.

8. linear test

Precision takes by weighing Bergeninum reference substance 13.22mg, puts in the 50ml measuring bottle, adds methanol and is diluted to scale, shakes up; Precision measures 0.5,1,2,3,5,10ml, puts respectively in the 25ml measuring bottle, adds methanol and is diluted to scale; Shake up, precision is measured above-mentioned solution 10 μ l respectively, injects chromatograph of liquid.With concentration is abscissa, is vertical coordinate with the peak area, carries out linear regression.Getting regression equation is: A=14656C-8375, coefficient R=0.9995.The result shows that Bergeninum linear relationship of concentration and peak area in 5.29～105.76 μ g/ml concentration ranges is good.

9. recovery test

Precision takes by weighing Bergeninum reference substance 12.5mg, puts in the 100ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, as the reference substance solution stock solution.

The reference substance solution precision is measured reference substance solution stock solution 10ml, puts in the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets.

The recovery test need testing solution is got the sample of measuring content in advance, and porphyrize takes by weighing about 0.25g, and accurate the title decides; The accurate reference substance solution 5ml that adds, the accurate methanol 5ml that adds claims to decide weight, supersound process (power 200W; Frequency 40kHz) 30 minutes, put coldly, claim again to decide weight, supply the weight that subtracts mistake with methanol; Shake up, filter, get subsequent filtrate, promptly get.Prepare 6 parts altogether.

Precision is measured response rate need testing solution and each 10 μ l of contrast solution respectively, injects chromatograph of liquid respectively.

Table 3 Bergeninum assay recovery test result

Can know that by result of the test this law recovery test accuracy is higher.

10. precision test

(1) assay replica test

Get lot number and be 20090222 the peaceful particulate samples of peace youngster, according to the method under the assay item, replication 6 times, the result sees the following form.

Table 4 Bergeninum assay replica test result

Conclusion: this product replica test is good.

(2) precision test in the middle of

Get lot number and be 20090222 the peaceful particulate samples of peace youngster, the method according under the assay item, adopt same Equipment Inspection by different analysts at different time respectively, result such as following table:

Precision test result in the middle of the table 5 Bergeninum assay

11. ruggedness

Get lot number and be 20090222 need testing solution, room temperature (25 ± 2 ℃) is placed, and measures 10 μ l respectively at 0,2,4,6,8 hour precision, injects chromatograph of liquid respectively.

Table 6 Bergeninum solution stability testing result

Conclusion: the peaceful granule of peace youngster is placed to measure in 8 hours in room temperature (25 ± 2 ℃) and is stablized.

12. sample size is measured

Get 3 batches of the peaceful granules of peace youngster, measure and calculate glycyrrhizic acid content, result such as following table.

Table 7 sample size is measured the result

Following embodiment all can realize the described effect of above-mentioned experimental example.

The specific embodiment

Embodiment 1: the particulate preparation of An Erning

The peaceful particulate crude drug of peace youngster consists of:

Flos Carthami 53.3 gram Santalum albums 66.7 grams

Radix Glycyrrhizae 53.3 gram high mountain Radix Cochleariae officinalis dishes 53.3 grams

Rhizoma Seu Herba Bergeniae 53.3 gram Concretio Silicea Bambusaes 66.7 grams

Lagotis brachystachya Maxim 66.7 gram Tangut aconite 66.7 grams

Artificial Calculus Bovis's 5.3 gram sucrose 930 grams.

The peaceful granule of peace youngster is prepared from following method:

Get Flos Carthami, Santalum album and add 5 times of amounts and extracted 3 hours, collect volatile oil, filter; Medicinal residues and Radix Glycyrrhizae, high mountain Radix Cochleariae officinalis dish, Rhizoma Seu Herba Bergeniae, Concretio Silicea Bambusae, short tube rabbit grass and Tangut aconite merge, and decocte with water 2 times adds 10 times of water gagings for the first time and decocted 2 hours; Add for the second time 8 times of water gagings and decocted 2 hours, filter merging filtrate; Be concentrated into the clear paste of ρ=1.02～1.04 (50 ℃ of mensuration of temperature), carry out cold preservation 24 hours in clear paste to the cold-storage jar, it is centrifugal with the high speed tube centrifuge to get supernatant; Centrifugal liquid continues to be concentrated into thick paste ρ=1.07～1.09 (50 ℃ of mensuration of temperature), and spray drying promptly gets and pacifies the peaceful extract of youngster;

Get beta-schardinger dextrin-10 grams; Be dissolved in 40 ℃ of water of 100 parts by volume; Ultrasonic 2 hours, under powerful magnetic agitation, add the volatile oil ethanolic solution of 2 parts by volume, contain the volatile oil of 0.5 parts by volume in wherein every parts by volume solution; The powerful stirring 2 hours; Refrigerate 24 hours, filter, 40 ℃ of vacuum drying precipitations; Pulverize, promptly get the Benexate Hydrochloride of volatile oil;

Get the peaceful extract of peace youngster, beta-schardinger dextrin-volatile oil clathrate compound and artificial Calculus Bovis, mix and add sucrose, mix homogeneously is done wetting agent with 50% alcoholic solution, and 14 orders are granulated, 50 ℃ of dryings, and 14 order granulate promptly get and pacify peaceful granule 1000 grams of youngster, every bag of 3g.

Embodiment 2: the peaceful particulate quality determining method of embodiment 1 said peace youngster

Differentiate:

A. get the peaceful granule 2g of peace youngster, porphyrize adds acetone 10ml, and supersound process 20 minutes filters, and filtrating is concentrated into 1ml, as need testing solution; Other gets cholic acid, Hyodeoxycholic Acid reference substance, adds acetone and processes the mixed solution that every 1ml contains 0.2mg, as reference substance solution; According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With E-C-glacial acetic acid (2: 2: 1) is developing solvent, launches, and takes out; Dry; Spray, is put under the ultra-violet lamp (365nm) and is inspected 105 ℃ of heating several minutes with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

B. after pacifying the peaceful granule porphyrize of youngster, get powder 10g, add methanol 20ml, supersound extraction 30min takes out; Put coldly, filter, get the subsequent filtrate evaporate to dryness after, residue water 20ml all is transferred to separatory funnel; Aqueous solution is with ethyl acetate extraction 4 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Other gets the Bergeninum reference substance, adds dissolve with methanol and be diluted to every 1ml methanol solution to contain the 0.2mg reference substance solution, shakes up, and compares article solution; According to the thin layer chromatography test, draw each 10 μ l of above-mentioned solution, put respectively on same silica GF254 lamellae, be developing solvent with chloroform-ethyl acetate-methanol (5: 4: 2.5), launch, taking-up is dried, and puts under the ultra-violet lamp 254nm and inspects; Be in the test sample chromatograph on the corresponding position of reference substance, show the speckle of same color;

Assay:

Embodiment 3: the peaceful particulate quality determining method of embodiment 1 said peace youngster

Differentiate:

Embodiment 4: the peaceful particulate quality determining method of embodiment 1 said peace youngster

Assay:

Embodiment 5: the peaceful particulate quality determining method of embodiment 1 said peace youngster

Differentiate:

Get the peaceful granule 2g of peace youngster, porphyrize adds acetone 10ml, and supersound process 20 minutes filters, and filtrating is concentrated into 1ml, as need testing solution; Other gets cholic acid, Hyodeoxycholic Acid reference substance, adds acetone and processes the mixed solution that every 1ml contains 0.2mg, as reference substance solution; According to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With E-C-glacial acetic acid (2: 2: 1) is developing solvent, launches, and takes out; Dry; Spray, is put under the ultra-violet lamp (365nm) and is inspected 105 ℃ of heating several minutes with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

Assay:

B. Bergeninum is measured according to high-efficient liquid phase technique (an appendix VI of Chinese Pharmacopoeia version in 2010 D);

Embodiment 6: the peaceful particulate quality determining method of embodiment 1 said peace youngster

Differentiate:

Behind the peaceful granule porphyrize of peace youngster, get powder 10g, add methanol 20ml, supersound extraction 30min takes out; Put coldly, filter, get the subsequent filtrate evaporate to dryness after, residue water 20ml all is transferred to separatory funnel; Aqueous solution is with ethyl acetate extraction 4 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Other gets the Bergeninum reference substance, adds dissolve with methanol and be diluted to every 1ml methanol solution to contain the 0.2mg reference substance solution, shakes up, and compares article solution; According to the thin layer chromatography test, draw each 10 μ l of above-mentioned solution, put respectively on same silica GF254 lamellae, be developing solvent with chloroform-ethyl acetate-methanol (5: 4: 2.5), launch, taking-up is dried, and puts under the ultra-violet lamp 254nm and inspects; Be in the test sample chromatograph on the corresponding position of reference substance, show the speckle of same color;

Assay:

A. glycyrrhizic acid is measured according to high-efficient liquid phase technique (an appendix VI of Chinese Pharmacopoeia version in 2010 D);

Embodiment 7: the peaceful particulate quality determining method of embodiment 1 said peace youngster

Differentiate:

Assay:

Claims

1. pacify the peaceful particulate quality determining method of youngster for one kind, it is characterized in that this method comprises one or more in following discriminating and/or the content assaying method:

Differentiate:

A. get the peaceful granule 1-3g of peace youngster, porphyrize adds acetone 5-15ml, and supersound process 10-30 minute, filter, filtrating is concentrated into 1ml, as need testing solution; Other gets cholic acid, Hyodeoxycholic Acid reference substance, adds acetone and processes the mixed solution that every 1ml contains 0.1-0.3mg, as reference substance solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2000) test, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With E-C-glacial acetic acid (1-3: 1-3: 0.5-1.5) be developing solvent, launch, take out; Dry; Spray, is put under the ultra-violet lamp (365nm) and is inspected 100 ℃-110 ℃ heating several minutes with 10% ethanol solution of sulfuric acid; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;

B. after pacifying the peaceful granule porphyrize of youngster, get powder 5-15g, add methanol 15-25ml, supersound extraction 20-40min; Take out, put coldly, filter; After getting the subsequent filtrate evaporate to dryness, residue water 10-30ml all is transferred to separatory funnel, and aqueous solution is with ethyl acetate extraction 3-5 time; Each 10-30ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methanol makes dissolving, is transferred in the 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Other gets the Bergeninum reference substance, adds dissolve with methanol and be diluted to every 1ml methanol solution to contain the 0.1-0.3mg reference substance solution, shakes up, and compares article solution; According to the thin layer chromatography test, draw each 10 μ l of above-mentioned solution, put respectively on same silica GF254 lamellae, with chloroform-ethyl acetate-methanol (3-7: 2-6: 1-4) be developing solvent, launch that taking-up is dried, and puts under the ultra-violet lamp 254nm and inspects; Be in the test sample chromatograph on the corresponding position of reference substance, show the speckle of same color;

Assay:

2. the peaceful particulate quality determining method of peace youngster as claimed in claim 1 is characterized in that this method comprises one or more in following discriminating and/or the content assaying method:

Differentiate:

Assay:

3. according to claim 1 or claim 2 quality determining method is characterized in that the peaceful particulate crude drug of wherein said peace youngster consists of:

Flos Carthami 30-70 weight portion Santalum album 40-70 weight portion

Artificial Calculus Bovis 2-8 weight portion sucrose 600-1200 weight portion.

4. quality determining method as claimed in claim 3 is characterized in that the peaceful particulate crude drug of wherein said peace youngster consists of:

Flos Carthami 53.3 weight portion Santalum albums 66.7 weight portions

Artificial Calculus Bovis's 5.3 weight portion sucrose 930 weight portions.

5. quality determining method as claimed in claim 3 is characterized in that the peaceful granule of wherein said peace youngster is prepared from following method:

Get the water that Flos Carthami, Santalum album add the 3-7 times of weight and extracted 2-4 hour, collect volatile oil, filter with steam distillation; Medicinal residues and Radix Glycyrrhizae, high mountain Radix Cochleariae officinalis dish, Rhizoma Seu Herba Bergeniae, Concretio Silicea Bambusae, short tube rabbit grass and Tangut aconite merge, add the 6-12 times of weight water, decoct 1-4 time; Time is 1-4 hour, filters merging filtrate; Be concentrated into 50 ℃ of clear paste of measuring ρ=1.02～1.04 down, carried out in clear paste to the cold-storage jar cold preservation 12-36 hour, it is centrifugal with the high speed tube centrifuge to get supernatant; Centrifugal liquid continues to be concentrated into 50 ℃ of thick pastes of measuring ρ=1.07～1.09 down, and spray drying promptly gets and pacifies the peaceful extract of youngster;

6. quality determining method as claimed in claim 4 is characterized in that the peaceful granule of wherein said peace youngster is prepared from following method:

7. like claim 5 or 6 described quality determining methods, it is characterized in that the peaceful granule of wherein said peace youngster is prepared from following method:

Get the water that Flos Carthami, Santalum album add 5 times of weight and extracted 3 hours, collect volatile oil, filter with steam distillation; Medicinal residues and Radix Glycyrrhizae, high mountain Radix Cochleariae officinalis dish, Rhizoma Seu Herba Bergeniae, Concretio Silicea Bambusae, short tube rabbit grass and Tangut aconite merge, decocte with water 2 times, and the decocting that adds 10 times of weight for the first time boiled 2 hours; The decocting that adds for the second time 8 times of weight boiled 2 hours, filtered merging filtrate; Be concentrated into 50 ℃ of clear paste of measuring ρ=1.02～1.04 down, carry out cold preservation 24 hours in clear paste to the cold-storage jar, it is centrifugal with the high speed tube centrifuge to get supernatant; Centrifugal liquid continues to be concentrated into 50 ℃ of thick pastes of measuring ρ=1.07～1.09 down, and spray drying promptly gets and pacifies the peaceful extract of youngster;