CN102579861B

CN102579861B - Method for detecting quality of An'erning granules

Info

Publication number: CN102579861B
Application number: CN201110009435.9A
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: 2014-11-05
Anticipated expiration: 2031-01-17
Also published as: CN102579861A

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

A kind of detection method of pacifying the peaceful particle of youngster

Technical field

The present invention relates to a kind of quality determining method of pharmaceutical composition, particularly the quality determining method of the peaceful particle of peace youngster.

Background technology

The peaceful particle of peace youngster is recorded in the national drug standards, standard number: WS-10640 (ZD-0640)-2002.There is removing heat to expel wind, the effect of preventing phlegm from forming and stopping coughing.For children's wind-heat cold, bring up phlegm when one coughs, heating pharyngalgia, the infection of the upper respiratory tract is shown in above-mentioned patient.

Children's upper respiratory tract infection, catch a cold, cough that to breathe heavily be common disease, frequently-occurring disease clinically, methods for the treatment of is a lot of, but effect differs.The peaceful particle of peace youngster is Tibetan medicine's traditional ancient proved recipe, and through Tibetan medicine's long-term clinical application, this medical instrument has the effect of clearing heat and detoxicating dispelling pathogenic wind and eliminating phlegm, for diseases such as children's's cold, pneumonia, bronchitis and the cough causing and heatings.

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

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

Summary of the invention

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

The present invention seeks to be achieved by the following scheme.

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

Differentiate:

A. get the peaceful particle 1-3g of peace youngster, porphyrize, adds acetone 5-15ml, and ultrasonic processing 10-30 minute filters, and filtrate is concentrated into 1ml, as need testing solution; Separately get cholic acid, hyodesoxycholic acid reference substance, add acetone and make every 1ml respectively containing the mixed solution of 0.1-0.3mg, in contrast product solution; According to thin-layered 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, take E-C-glacial acetic acid (1-3: 1-3: 0.5-1.5) be developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, 100 ℃-110 ℃ heating several minutes, is put under ultraviolet lamp (365nm) and is inspected; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

B. pacify after the peaceful particle porphyrize of youngster, get powder 5-15g, add methyl alcohol 15-25ml, ultrasonic extraction 20-40min, take out, let cool, filter, get after subsequent filtrate evaporate to dryness, residue water 10-30ml is all transferred to separating funnel, and aqueous solution is extracted 3-5 time with ethyl acetate, each 10-30ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methyl alcohol to be made to dissolve, and is transferred in 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Separately get Bergenin reference substance, add methyl alcohol and dissolve and be diluted to every 1ml methanol solution containing 0.1-0.3mg reference substance solution, shake up, compare product solution; According to thin-layered chromatography test, draw each 10 μ l of above-mentioned solution, put respectively on same silica GF254 thin layer plate, take methenyl choloride-ethyl acetate-methyl alcohol (3-7: 2-6: 1-4) be developping agent, expansion, taking-up is dried, and puts under ultraviolet lamp 254nm and inspects; In test sample chromatogram, be on the corresponding position of reference substance the spot of aobvious same color.

Assay

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

Chromatographic condition and system suitability: its silane group silica gel of octadecane of take is filling agent; Take methyl alcohol-tetrahydrofuran-0.5% acetum (70-90: 5-15: 5-15) be mobile phase; Detection wavelength is 450nm; Number of theoretical plate calculates and should be not less than 2000 by cholerythrin peak;

The preparation of reference substance solution: precision takes cholerythrin reference substance, chlorination is copied into every 1ml containing the solution of 5-15 μ g, obtains;

The preparation of need testing solution: after the peaceful particle porphyrize of peace youngster, get powder 3-7g, accurately weighed, to put in the conical flask of tool plug, precision adds methylene chloride 20-30ml, close plug, weighed weight, ultrasonic 20-40min, lets cool, the weight of supplying less loss with methylene chloride, shakes up, and filters, and obtains;

Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and injection liquid chromatography, measures, and obtains;

Pacify every gram of the peaceful particle of youngster and contain cholerythrin (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 (appendix VI D of Chinese Pharmacopoeia version in 2010);

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

The preparation of reference substance solution: precision takes ammonium glycyrrhetate reference substance, adds methyl alcohol and makes every 1ml containing the solution of 0.1-0.3mg, obtains (glycyrrhizic acid weight=ammonium glycyrrhetate/1.0207);

The preparation of need testing solution: after the peaceful particle porphyrize of peace youngster, get powder 5-8g, accurately weighed, to put in the conical flask of tool plug, precision adds methyl alcohol 20-30ml, close plug, weighed weight, ultrasonic 20-40min, lets cool, the weight of supplying less loss with methyl alcohol, shakes up, and filters, and obtains;

Pacify every gram of the peaceful particle of youngster and contain glycyrrhizic acid (C ₄₂h ₆₁o ₁₆nH ₄) meter, must not be less than 0.33mg;

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

Chromatographic condition and system suitability: its silane group silica gel of octadecane of take is filling agent; Take methanol-water (10-30: 70-90) be mobile phase; Detection wavelength is 275nm; Number of theoretical plate calculates and should be not less than 2000 by Bergenin peak;

The preparation of reference substance solution: precision takes Bergenin reference substance, adds methyl alcohol and makes every 1ml containing the solution of 40-60 μ g, obtains;

The preparation of need testing solution: after the peaceful particle porphyrize of peace youngster, get powder 2-4g, accurately weighed, to put in the conical flask of tool plug, precision adds methyl alcohol 40-60ml, close plug, weighed weight, ultrasonic 20-40min, lets cool, the weight of supplying less loss with methyl alcohol, shakes up, and filters, and obtains;

Pacify every gram of the peaceful particle of youngster and contain Bergenin (C ₁₄h ₁₆o ₉) meter, must not be less than 0.67mg.

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

Differentiate:

A. get the peaceful particle 2g of peace youngster, porphyrize, adds acetone 10ml, and ultrasonic processing 20 minutes filters, and filtrate is concentrated into 1ml, as need testing solution; Separately get cholic acid, hyodesoxycholic acid reference substance, add acetone and make every 1ml respectively containing the mixed solution of 0.2mg, in contrast product solution; According to thin-layered chromatography (appendix VI B 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, E-C-the glacial acetic acid (2: 2: 1) of take is developping agent, launches, and takes out, dry, spray, with 10% ethanol solution of sulfuric acid, 105 ℃ of heating several minutes, is put under ultraviolet lamp (365nm) and is inspected; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color.

B. pacify after the peaceful particle porphyrize of youngster, get powder 10g, add methyl alcohol 20ml, ultrasonic extraction 30min, takes out, let cool, filter, get after subsequent filtrate evaporate to dryness, residue water 20ml is all transferred to separating funnel, aqueous solution is extracted 4 times with ethyl acetate, each 20ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methyl alcohol to be made to dissolve, and is transferred in 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Separately get Bergenin reference substance, add methyl alcohol and dissolve and be diluted to every 1ml methanol solution containing 0.2mg reference substance solution, shake up, compare product solution; According to thin-layered chromatography test, draw each 10 μ l of above-mentioned solution, to put respectively on same silica GF254 thin layer plate, the methenyl choloride-ethyl acetate-methyl alcohol (5: 4: 2.5) of take is developping agent, launches, taking-up is dried, and puts under ultraviolet lamp 254nm and inspects; In test sample chromatogram, be on the corresponding position of reference substance the spot of aobvious same color.

Assay:

Chromatographic condition and system suitability: its silane group silica gel of octadecane of take is filling agent; Methyl alcohol-tetrahydrofuran-0.5% acetum (80: 10: 10) of take is mobile phase; Detection wavelength is 450nm; Number of theoretical plate calculates and should be not less than 2000 by cholerythrin peak;

The preparation of reference substance solution: precision takes cholerythrin reference substance, chlorination is copied into every 1ml containing the solution of 10 μ g, obtains;

The preparation of need testing solution: after the peaceful particle porphyrize of peace youngster, get powder 5g, accurately weighed, to put in the conical flask of tool plug, precision adds methylene chloride 25ml, close plug, weighed weight, ultrasonic 30min, lets cool, the weight of supplying less loss with methylene chloride, shakes up, and filters, and obtains;

Pacify every gram of the peaceful particle of youngster and contain cholerythrin (C ₃₃h ₃₆n ₄o ₆) meter, must not be less than 0.04mg.

Chromatographic condition and system suitability: its silane group silica gel of octadecane of take is filling agent; Acetonitrile-0.5% phosphoric acid solution (37: 63) of take is mobile phase; Detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 5000 by Radix Glycyrrhizae acid amide peak;

The preparation of reference substance solution: precision takes ammonium glycyrrhetate reference substance, adds methyl alcohol and makes every 1ml containing the solution of 0.2mg, obtains (glycyrrhizic acid weight=ammonium glycyrrhetate/1.0207);

The preparation of need testing solution: after the peaceful particle porphyrize of peace youngster, get powder 6.5g, accurately weighed, to put in the conical flask of tool plug, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic 30min, lets cool, and supplies the weight of less loss with methyl alcohol, shakes up, and filters, and obtains;

Pacify every gram of the peaceful particle of youngster and contain glycyrrhizic acid (C ₄₂h ₆₁o ₁₆nH ₄) meter, must not be less than 0.33mg.

Chromatographic condition and system suitability: its silane group silica gel of octadecane of take is filling agent; The methanol-water (20: 80) of take is mobile phase; Detection wavelength is 275nm; Number of theoretical plate calculates and should be not less than 2000 by Bergenin peak;

The preparation of reference substance solution: precision takes Bergenin reference substance, adds methyl alcohol and makes every 1ml containing the solution of 50 μ g, obtains;

The preparation of need testing solution: after the peaceful particle porphyrize of peace youngster, get powder 3g, accurately weighed, to put in the conical flask of tool plug, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic 30min, lets cool, and supplies the weight of less loss with methyl alcohol, shakes up, and filters, and obtains;

Wherein, the bulk drug of the peaceful particle of above-mentioned peace youngster consists of:

Safflower 30-70 weight portion Santalum album 40-70 weight portion

Radix Glycyrrhizae 30-70 weight portion high mountain horseradish dish 30-70 weight portion

Purple bergenia herb 30-70 weight portion Tabasheer 40-70 weight portion

Lagotis brevituba 40-70 weight portion Tangut aconite 40-70 weight portion

Calculus bovis factitius 2-8 weight portion sucrose 600-1200 weight portion.

Wherein, the bulk drug of the peaceful particle of above-mentioned peace youngster composition is preferably:

Safflower 53.3 weight portion Santalum album 66.7 weight portions

Radix Glycyrrhizae 53.3 weight portion high mountain horseradish dish 53.3 weight portions

Purple bergenia herb 53.3 weight portion Tabasheer 66.7 weight portions

Lagotis brachystachya Maxim 66.7 weight portion Tangut aconite 66.7 weight portions

Calculus bovis factitius 5.3 weight portion sucrose 930 weight portions.

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

Get safflower, the water that Santalum album adds 3-7 times of weight extracts 2-4 hour with steam distillation, collect volatile oil, filter, the dregs of a decoction and Radix Glycyrrhizae, high mountain horseradish dish, purple bergenia herb, Tabasheer, Lagotis brevituba and Tangut aconite merge, add 6-12 times of weight water, decoct 1-4 time, time is 1-4 hour, filter, merging filtrate, be concentrated into the clear cream of ρ=1.02～1.04 (temperature 50 C mensuration), clear cream refrigerates 12-36 hour to cold-storage jar, get supernatant centrifugal with high speed tube centrifuge, centrifugate continues to be concentrated into thick paste ρ=1.07～1.09 (temperature 50 C mensuration), spraying is dry, must pacify 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 adds the volatile oil ethanolic solution of 1-3 parts by volume under powerful magnetic agitation, wherein in every parts by volume solution containing the volatile oil of 0.3-0.8 parts by volume, strong stirring 1-6 hour, refrigeration 12-36 hour, filters, 30-50 ℃ of vacuum drying precipitation, pulverize, obtain the Benexate Hydrochloride of volatile oil;

Get the peaceful extract of peace youngster, beta-schardinger dextrin-volatile oil clathrate compound and calculus bovis factitius, mix and add sucrose, mix, with 50% ethanolic solution, make wetting agent, 14 orders are granulated, and 50 ℃ dry, and the whole grain of 14 orders, obtains.

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

Get safflower, the water that Santalum album adds 5 times of weight extracts 3 hours with steam distillation, collect volatile oil, filter, the dregs of a decoction and Radix Glycyrrhizae, high mountain horseradish dish, purple bergenia herb, Tabasheer, Lagotis brevituba and Tangut aconite merge, boiling 2 times, the decocting that adds for the first time 10 times of weight boils 2 hours, the decocting that adds for the second time 8 times of weight boils 2 hours, filter, merging filtrate, be concentrated into the clear cream of ρ=1.02～1.04 (temperature 50 C mensuration), clear cream refrigerates 24 hours to cold-storage jar, get supernatant centrifugal with high speed tube centrifuge, centrifugate continues to be concentrated into thick paste ρ=1.07～1.09 (temperature 50 C mensuration), spraying is dry, must pacify 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, wherein in every parts by volume solution containing the volatile oil of 0.5 parts by volume, strong stirring 2 hours, refrigerate 24 hours, filter, 40 ℃ of vacuum drying precipitations, pulverize, obtain the Benexate Hydrochloride of volatile oil;

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

The invention discloses a kind of quality determining method about the peaceful particle of peace youngster, the method comprises the discrimination method of cholic acid and Bergenin, also comprises glycyrrhizic acid, Bergenin and bilirubinic content assaying method.Through experimental study, the discrimination method in quality determining method of the present invention in the test sample chromatogram of sample, with the corresponding position of reference substance chromatogram on, equal spots of aobvious same color; Negative control is noiseless.With the glycyrrhizic acid in the peaceful particle of content assaying method mensuration peace youngster of the present invention and the content of Bergenin, result shows, the method linear relationship is good, and recovery test accuracy is higher, there is fabulous repeatability and stability, can control accurately, closely the quality of pacifying the peaceful particle of youngster.

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

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

According to being recorded in the national drug standards, standard number is: WS-10640 (ZD-0640)-2002, according to the metric system of the peaceful particle of peace youngster, for three batches of peaceful particles of peace youngster, respectively the discrimination method of wherein each kind of bulk drug is tested, experimental basis and result are as follows:

1, with reference to Quality of Safflower standard safflower test of < < Chinese Pharmacopoeia > > version in 2010, result shows in three batch samples and negative control chromatogram, with the corresponding position of control medicinal material chromatogram on, without obvious spot.Therefore do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

2, the tlc identification method > > with reference to < < compound bergenin pills tests purple bergenia herb, result shows in three batch samples and negative control chromatogram, with the corresponding position of reference substance chromatogram on, equal spots of aobvious same color; Negative control has interference.Therefore do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

3, the content > > with reference to < < determination of arolisic acid B in Qingfei Xiaoyan Pills with Thin-layer chromatography scanning method tests purple bergenia herb, result shows in three batches of test sample chromatograms, with the corresponding position of reference substance chromatogram on, equal spots of aobvious same color; Negative control is noiseless.Pretend and pacify the part in the peaceful granular mass detection method of youngster for the present invention.

4, with reference to < < Lagotis glauca medicinal material thin-layer chromatography, differentiate with assay > > Lagotis brachystachya Maxim is tested, result shows in three batch samples and negative control chromatogram, with the corresponding position of reference substance chromatogram on, equal spots of aobvious same color; Negative control has interference.Therefore do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

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

6, the discriminating > > with reference to ingredient Catalpol in < < glutinous rehmannia beverage tests Lagotis brachystachya Maxim, result shows in three batch samples and negative control chromatogram, with the corresponding position of reference substance chromatogram on, equal spots of aobvious same color; Negative control has interference.Therefore do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

7, with reference to Tabasheer quality standard of < < Chinese Pharmacopoeia > > version in 2010, Tabasheer is tested, result shows that feminine gender has interference.Therefore 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, Radix Glycyrrhizae is tested, result shows to have no ammonium glycyrrhetate obvious spot, the aobvious spot of Radix Glycyrrhizae control medicinal material, also there is identical spot test sample and negative control corresponding position, therefore feminine gender has interference.Therefore do not pacify the part in the peaceful granular mass detection method of youngster as the present invention.

9, with reference to the thin layer of Radix Glycyrrhizae in < < brown mixture, differentiate that > > tests Radix Glycyrrhizae, result shows that test sample and negative control also have identical spot in ammonium glycyrrhetate corresponding position, therefore feminine gender has interference.Therefore 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 particle of peace youngster

1. instrument, reagent and test sample

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

Reference substance: ammonium glycyrrhetate reference substance

Sample: the peace peaceful particle of youngster (standby according to the metric system that is recorded in the peaceful particle of peace youngster in Chinese patent drug provincial standard rising national drug standards part)

2. the selection of mobile phase

With reference to Radix Glycyrrhizae assay item, intending selecting acetonitrile-0.5% phosphoric acid solution (37: 63) is mobile phase, and with this understanding, ammonium glycyrrhetate and other adjacent peak degree of separation are larger, and peak shape is better.

3. detect the selection of wavelength

Extracting Radix Glycyrrhizae acid ammonium reference substance solution scans in 200-400nm wavelength coverage, and according to ultraviolet absorpting spectrum, selected 250nm is for detecting wavelength.

4. the preparation of reference substance solution

With reference to Radix Glycyrrhizae content assaying method, determine that the preparation method of reference substance solution is: it is appropriate that precision takes ammonium glycyrrhetate reference substance, add 70% ethanol and make every 1ml containing the solution of 0.2mg and get final product.(glycyrrhizic acid weight=ammonium glycyrrhetate weight/1.0207)

5. the preparation of need testing solution

Get powder 4.0g after peace youngster peaceful particle porphyrize, accurately weighed, to put in conical flask, precision adds 70% ethanol 25ml, weighed weight, ultrasonic processing 30min, after letting cool, supplies the weight of less loss with 70% ethanol, shake up, and filtration, obtains.

6. system suitability and the negative investigation of disturbing

Under above-mentioned chromatographic condition, precision is drawn each 10 μ l of blank solvent, reference substance solution, need testing solution and negative control solution respectively, injection liquid chromatography, and result shows, the degree of separation that in test sample chromatogram, ammonium glycyrrhetate is adjacent chromatographic peak is greater than 1.5, and negative control is noiseless.

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

Precision measures ammonium glycyrrhetate reference substance stock solution solution (0.3614mg/ml) 1ml, 2ml, 3ml, 5ml, 8ml, 10ml, put respectively in 10ml volumetric flask, 70% ethanol is diluted to scale, shake up, each accurate sample introduction 10 μ l, with peak area (A), reference substance concentration (C) is carried out to linear regression, obtain regression equation: A=7175.9C+43512, related coefficient: R=0.9993.Result shows: within the scope of 36.14ug/ml～361.40ug/ml, the peak area of ammonium glycyrrhetate (A) is good with concentration (C) linear relationship.

8. precision test

Precision is drawn ammonium glycyrrhetate reference substance solution 10 μ l respectively, injection liquid chromatography, and each METHOD FOR CONTINUOUS DETERMINATION 6 times, records peak area and calculates relative standard deviation, RSD=0.80%.Result shows, instrument precision is good.

9. stability test

After prepared by need testing solution, the accurate 10 μ l that draw, injection liquid chromatography, records peak area, measures once every 2 hours later, investigates 8 hours, calculates the relative standard deviation of peak area, RSD=0.90%.Result shows: test sample mesobilirubin measurement result in 8 hours is stable.

10. replica test

Get the peaceful particle of peace youngster, replication 6 times, the content of glycyrrhizic acid in calculation sample.Result glycyrrhizic acid average content is: 0.43mg/g, RSD=0.99%.Show that analytical approach repeatability is good.

11. recovery tests

Precision takes with 6 parts of the peaceful particulate samples of a collection of peace youngster, and precision adds ammonium glycyrrhetate reference substance, measures its content, calculate recovery rate, and result is as follows.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 determinations

Get 3 batches of the peaceful particles of peace youngster, measure and calculate glycyrrhizic acid content, result is as follows.Table 2

The peaceful particulate samples assay of table 2 peace youngster result

Experimental example 3HPLC method is measured the content of Bergenin in the peaceful particle of peace youngster

1. instrument, reagent and test sample

Reference substance: Bergenin reference substance

2. mobile phase is selected

With reference to Determination of Bergenin in ardisia japonica quality standard of < < Chinese Pharmacopoeia > > version in 2005, measure a lower method test, intending selecting methanol-water (20: 80) is mobile phase.

Get Bergenin reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 50 μ g, obtain.

3, the preparation of purple bergenia herb blank solution

By prescription preparation, do not contain the negative control of purple bergenia herb, with need testing solution operational processes.

Under this chromatographic condition, precision measures each 10 μ l of above-mentioned solution, difference injection liquid chromatography, and result Bergenin main peak retention time is 13.257min, number of theoretical plate is 6428.Negative sample is noiseless.

4. detecting wavelength determines

Get Bergenin reference substance appropriate, accurately weighed, add methyl alcohol dissolving and quantitatively dilute and make the solution that approximately contains 20 μ g in every 1ml, in 200～400nm wavelength coverage, carry out UV scanning, result shows that Bergenin has absorption maximum at 273.4nm wavelength place, with reference to content assaying method in ardisia japonica quality standard of < < Chinese Pharmacopoeia > > version in 2010, determine that it is 275nm that this product assay detects wavelength simultaneously.

5. the preparation of reference substance solution

6. the preparation of need testing solution

After the peaceful particle porphyrize of peace youngster, get powder 3g, accurately weighed, to put in the conical flask of tool plug, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic 30min, lets cool, and supplies the weight of less loss with methyl alcohol, shakes up, and filters, and obtains.

7. system suitability and the negative investigation of disturbing

Under above-mentioned chromatographic condition, precision is drawn each 10 μ l of blank solvent, reference substance solution, need testing solution and negative control solution respectively, injection liquid chromatography, and result shows, the degree of separation that in test sample chromatogram, Bergenin is adjacent chromatographic peak is greater than 1.5, and negative control is noiseless.

8. linear test

Precision takes Bergenin reference substance 13.22mg, puts in 50ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, precision measures 0.5,1,2,3,5,10ml, puts respectively in 25ml measuring bottle, adds methyl alcohol and is diluted to scale, shake up, precision measures above-mentioned solution 10 μ l, injection liquid chromatography respectively.Take concentration as horizontal ordinate, take peak area as ordinate, carry out linear regression.Obtaining regression equation is: A=14656C-8375, coefficient R=0.9995.Result shows that Bergenin linear relationship of concentration and peak area in 5.29～105.76 μ g/ml concentration ranges is good.

9. recovery test

Precision takes Bergenin reference substance 12.5mg, puts in 100ml measuring bottle, adds methyl alcohol and dissolves and be diluted to scale, shakes up, in contrast product solution stock solution.

Reference substance solution precision measures reference substance solution stock solution 10ml, puts in 25ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and obtains.

Recovery test need testing solution is got the sample of measuring in advance content, and porphyrize takes about 0.25g, accurately weighed, precision adds reference substance solution 5ml, and precision adds methyl alcohol 5ml, weighed weight, ultrasonic processing (power 200W, frequency 40kHz) 30 minutes, let cool, more weighed weight, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate, obtain.Prepare altogether 6 parts.

Precision measures recovery need testing solution and each 10 μ l of contrast solution, respectively injection liquid chromatography respectively.

Table 3 Determination of Bergenin is measured recovery test result

As seen from the experiment, the accuracy of this law recovery test 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 assay item, replication 6 times, the results are shown in following table.

Table 4 Determination of Bergenin is measured 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 assay item, adopts same equipment Inspection by different analysts at different time respectively, and result is as following table:

Table 5 Determination of Bergenin is measured middle Precision test result

11. durabilities

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

Table 6 Bergenin solution stability testing result

Conclusion: the peaceful particle of peace youngster is placed in 8 hours and measured and stablize in room temperature (25 ± 2 ℃).

12. sample sizes are measured

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

Table 7 sample size measurement result

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

Embodiment

Embodiment 1: the preparation of the peaceful particle of peace youngster

The bulk drug of the peaceful particle of peace youngster consists of:

66.7 grams of 53.3 grams of Santalum albums of safflower

53.3 grams of 53.3 grams of high mountain horseradish dishes of Radix Glycyrrhizae

66.7 grams of 53.3 grams of Tabasheers of purple bergenia herb

66.7 grams of 66.7 grams of Tangut aconites of Lagotis brachystachya Maxim

930 grams of 5.3 grams of sucrose of calculus bovis factitius.

The peaceful particle of peace youngster is prepared from by the following method:

Get safflower, Santalum album adds 5 times of amounts and extracts 3 hours, collect volatile oil, filter, the dregs of a decoction and Radix Glycyrrhizae, high mountain horseradish dish, purple bergenia herb, Tabasheer, Lagotis brevituba and Tangut aconite merge, boiling 2 times, adding for the first time 10 times of water gagings decocts 2 hours, adding for the second time 8 times of water gagings decocts 2 hours, filter, merging filtrate, be concentrated into the clear cream of ρ=1.02～1.04 (temperature 50 C mensuration), clear cream refrigerates 24 hours to cold-storage jar, get supernatant centrifugal with high speed tube centrifuge, centrifugate continues to be concentrated into thick paste ρ=1.07～1.09 (temperature 50 C mensuration), spraying is dry, must pacify the peaceful extract of youngster,

Get 10 grams of beta-schardinger dextrin-s, 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, wherein in every parts by volume solution containing the volatile oil of 0.5 parts by volume, strong stirring 2 hours, refrigerate 24 hours, filter, 40 ℃ of vacuum drying precipitations, pulverize, obtain the Benexate Hydrochloride of volatile oil;

Get the peaceful extract of peace youngster, beta-schardinger dextrin-volatile oil clathrate compound and calculus bovis factitius, mix and add sucrose, mix, with 50% ethanolic solution, make wetting agent, 14 orders are granulated, and 50 ℃ dry, and the whole grain of 14 orders, must pacify 1000 grams of the peaceful particles of youngster, every bag of 3g.

Embodiment 2: the quality determining method of pacifying the peaceful particle of youngster described in embodiment 1

Differentiate:

A. get the peaceful particle 2g of peace youngster, porphyrize, adds acetone 10ml, and ultrasonic processing 20 minutes filters, and filtrate is concentrated into 1ml, as need testing solution; Separately get cholic acid, hyodesoxycholic acid reference substance, add acetone and make every 1ml respectively containing the mixed solution of 0.2mg, in contrast product solution; According to thin-layered chromatography (appendix VI B 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, E-C-the glacial acetic acid (2: 2: 1) of take is developping agent, launches, and takes out, dry, spray, with 10% ethanol solution of sulfuric acid, 105 ℃ of heating several minutes, is put under ultraviolet lamp (365nm) and is inspected; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

B. pacify after the peaceful particle porphyrize of youngster, get powder 10g, add methyl alcohol 20ml, ultrasonic extraction 30min, takes out, let cool, filter, get after subsequent filtrate evaporate to dryness, residue water 20ml is all transferred to separating funnel, aqueous solution is extracted 4 times with ethyl acetate, each 20ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methyl alcohol to be made to dissolve, and is transferred in 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Separately get Bergenin reference substance, add methyl alcohol and dissolve and be diluted to every 1ml methanol solution containing 0.2mg reference substance solution, shake up, compare product solution; According to thin-layered chromatography test, draw each 10 μ l of above-mentioned solution, to put respectively on same silica GF254 thin layer plate, the methenyl choloride-ethyl acetate-methyl alcohol (5: 4: 2.5) of take is developping agent, launches, taking-up is dried, and puts under ultraviolet lamp 254nm and inspects; In test sample chromatogram, be on the corresponding position of reference substance the spot of aobvious same color;

Assay:

Embodiment 3: the quality determining method of pacifying the peaceful particle of youngster described in embodiment 1

Differentiate:

Embodiment 4: the quality determining method of pacifying the peaceful particle of youngster described in embodiment 1

Assay:

Embodiment 5: the quality determining method of pacifying the peaceful particle of youngster described in embodiment 1

Differentiate:

Get the peaceful particle 2g of peace youngster, porphyrize, adds acetone 10ml, and ultrasonic processing 20 minutes filters, and filtrate is concentrated into 1ml, as need testing solution; Separately get cholic acid, hyodesoxycholic acid reference substance, add acetone and make every 1ml respectively containing the mixed solution of 0.2mg, in contrast product solution; According to thin-layered chromatography (appendix VI B 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, E-C-the glacial acetic acid (2: 2: 1) of take is developping agent, launches, and takes out, dry, spray, with 10% ethanol solution of sulfuric acid, 105 ℃ of heating several minutes, is put under ultraviolet lamp (365nm) and is inspected; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

Assay:

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

Embodiment 6: the quality determining method of pacifying the peaceful particle of youngster described in embodiment 1

Differentiate:

After the peaceful particle porphyrize of peace youngster, get powder 10g, add methyl alcohol 20ml, ultrasonic extraction 30min, takes out, let cool, filter, get after subsequent filtrate evaporate to dryness, residue water 20ml is all transferred to separating funnel, aqueous solution is extracted 4 times with ethyl acetate, each 20ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methyl alcohol to be made to dissolve, and is transferred in 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Separately get Bergenin reference substance, add methyl alcohol and dissolve and be diluted to every 1ml methanol solution containing 0.2mg reference substance solution, shake up, compare product solution; According to thin-layered chromatography test, draw each 10 μ l of above-mentioned solution, to put respectively on same silica GF254 thin layer plate, the methenyl choloride-ethyl acetate-methyl alcohol (5: 4: 2.5) of take is developping agent, launches, taking-up is dried, and puts under ultraviolet lamp 254nm and inspects; In test sample chromatogram, be on the corresponding position of reference substance the spot of aobvious same color;

Assay:

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

Embodiment 7: the quality determining method of pacifying the peaceful particle of youngster described in embodiment 1

Differentiate:

Assay:

Claims

1. pacify a detection method for the peaceful particle of youngster, it is characterized in that the method comprises following discriminating and assay:

Differentiate:

A. get the peaceful particle 1-3g of peace youngster, porphyrize, adds acetone 5-15ml, and ultrasonic processing 10-30 minute filters, and filtrate is concentrated into 1ml, as need testing solution; Separately get cholic acid, hyodesoxycholic acid reference substance, add acetone and make every 1ml respectively containing the mixed solution of 0.1-0.3mg, in contrast product solution; According to the thin-layered chromatography test of an appendix VI B of Chinese Pharmacopoeia version in 2000, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, E-C-glacial acetic acid that the volume ratio of take is 1-3:1-3:0.5-1.5 is developping agent, launches, and takes out, dry, spray, with 10% ethanol solution of sulfuric acid, 100 ℃-110 ℃ heating several minutes, is put under 365nm ultraviolet lamp and is inspected; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

B. pacify after the peaceful particle porphyrize of youngster, get powder 5-15g, add methyl alcohol 15-25ml, ultrasonic extraction 20-40min, take out, let cool, filter, get after subsequent filtrate evaporate to dryness, residue water 10-30ml is all transferred to separating funnel, and aqueous solution is extracted 3-5 time with ethyl acetate, each 10-30ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methyl alcohol to be made to dissolve, and is transferred in 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Separately get Bergenin reference substance, add methyl alcohol and dissolve and be diluted to every 1ml methanol solution containing 0.1-0.3mg reference substance solution, shake up, compare product solution; According to thin-layered chromatography test, draw each 10 μ l of above-mentioned solution, put respectively on same silica GF254 thin layer plate, methenyl choloride-ethyl acetate-methyl alcohol that the volume ratio of take is 3-7:2-6:1-4 is developping agent, launch, taking-up is dried, and puts under ultraviolet lamp 254nm and inspects; In test sample chromatogram, be on the corresponding position of reference substance the spot of aobvious same color;

Assay:

A. according to the Syrups by HPLC cholerythrin of an appendix VI D of Chinese Pharmacopoeia version in 2010;

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-tetrahydrofuran-0.5% acetum that the volume ratio of take is 70-90:5-15:5-15 is mobile phase; Detection wavelength is 450nm; Number of theoretical plate calculates and should be not less than 2000 by cholerythrin peak;

Pacify every gram of the peaceful particle of youngster and contain cholerythrin C ₃₃h ₃₆n ₄o ₆meter, must not be less than 0.04mg;

B. according to the Syrups by HPLC glycyrrhizic acid of an appendix VI D of Chinese Pharmacopoeia version in 2010;

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Acetonitrile-0.5% phosphoric acid solution that the volume ratio of take is 30-40:60-70 is mobile phase; Detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 5000 by Radix Glycyrrhizae acid amide peak;

The preparation of reference substance solution: precision takes ammonium glycyrrhetate reference substance, adds methyl alcohol and makes every 1ml containing the solution of 0.1-0.3mg, obtains, wherein glycyrrhizic acid weight=ammonium glycyrrhetate/1.0207;

Pacify every gram of the peaceful particle of youngster and contain glycyrrhizic acid C ₄₂h ₆₁o ₁₆nH ₄meter, must not be less than 0.33mg;

C. according to the Syrups by HPLC Bergenin of an appendix VI D of Chinese Pharmacopoeia version in 2010;

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; The methanol-water that the volume ratio of take is 10-30:70-90 is mobile phase; Detection wavelength is 275nm; Number of theoretical plate calculates and should be not less than 2000 by Bergenin peak;

Pacify every gram of the peaceful particle of youngster and contain Bergenin C ₁₄h ₁₆o ₉meter, must not be less than 0.67mg;

Wherein, the bulk drug of the peaceful particle of described peace youngster consists of:

2. the detection method of the peaceful particle of peace as claimed in claim 1 youngster, is characterized in that the method comprises following discriminating and assay:

Differentiate:

A. get the peaceful particle 2g of peace youngster, porphyrize, adds acetone 10ml, and ultrasonic processing 20 minutes filters, and filtrate is concentrated into 1ml, as need testing solution; Separately get cholic acid, hyodesoxycholic acid reference substance, add acetone and make every 1ml respectively containing the mixed solution of 0.2mg, in contrast product solution; According to the thin-layered chromatography test of an appendix VI B of Chinese Pharmacopoeia version in 2000, draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, E-C-glacial acetic acid that the volume ratio of take is 2:2:1 is developping agent, launches, and takes out, dry, spray, with 10% ethanol solution of sulfuric acid, 105 ℃ of heating several minutes, is put under 365nm ultraviolet lamp and is inspected; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the fluorescence spot of aobvious same color;

B. pacify after the peaceful particle porphyrize of youngster, get powder 10g, add methyl alcohol 20ml, ultrasonic extraction 30min, takes out, let cool, filter, get after subsequent filtrate evaporate to dryness, residue water 20ml is all transferred to separating funnel, aqueous solution is extracted 4 times with ethyl acetate, each 20ml, combined ethyl acetate liquid, evaporate to dryness; Residue adds methyl alcohol to be made to dissolve, and is transferred in 5ml measuring bottle, and is diluted to scale, shakes up, and makes need testing solution; Separately get Bergenin reference substance, add methyl alcohol and dissolve and be diluted to every 1ml methanol solution containing 0.2mg reference substance solution, shake up, compare product solution; According to thin-layered chromatography test, draw each 10 μ l of above-mentioned solution, to put respectively on same silica GF254 thin layer plate, methenyl choloride-ethyl acetate-methyl alcohol that the volume ratio of take is 5:4:2.5 is developping agent, launches, taking-up is dried, and puts under ultraviolet lamp 254nm and inspects; In test sample chromatogram, be on the corresponding position of reference substance the spot of aobvious same color;

Assay:

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-tetrahydrofuran-0.5% acetum that the volume ratio of take is 80:10:10 is mobile phase; Detection wavelength is 450nm; Number of theoretical plate calculates and should be not less than 2000 by cholerythrin peak;

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Acetonitrile-0.5% phosphoric acid solution that the volume ratio of take is 37:63 is mobile phase; Detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 5000 by Radix Glycyrrhizae acid amide peak;

The preparation of reference substance solution: precision takes ammonium glycyrrhetate reference substance, adds methyl alcohol and makes every 1ml containing the solution of 0.2mg, obtains, wherein glycyrrhizic acid weight=ammonium glycyrrhetate/1.0207;

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; The methanol-water that the volume ratio of take is 20:80 is mobile phase; Detection wavelength is 275nm; Number of theoretical plate calculates and should be not less than 2000 by Bergenin peak;

Pacify every gram of the peaceful particle of youngster and contain Bergenin C ₁₄h ₁₆o ₉meter, must not be less than 0.67mg.

3. detection method as claimed in claim 1 or 2, is characterized in that the peaceful particle of wherein said peace youngster is prepared from by the following method:

Get safflower, the water that Santalum album adds 3-7 times of weight extracts 2-4 hour with steam distillation, collect volatile oil, filter, the dregs of a decoction and Radix Glycyrrhizae, high mountain horseradish dish, purple bergenia herb, Tabasheer, Lagotis brevituba and Tangut aconite merge, add 6-12 times of weight water, decoct 1-4 time, time is 1-4 hour, filter, merging filtrate, be concentrated into the clear cream of measuring ρ=1.02～1.04 at 50 ℃, clear cream refrigerates 12-36 hour to cold-storage jar, get supernatant centrifugal with high speed tube centrifuge, centrifugate continues to be concentrated into the thick paste of measuring ρ=1.07～1.09 at 50 ℃, spraying is dry, must pacify the peaceful extract of youngster,

4. detection method as claimed in claim 3, is characterized in that the peaceful particle of wherein said peace youngster is prepared from by the following method:

Get safflower, the water that Santalum album adds 5 times of weight extracts 3 hours with steam distillation, collect volatile oil, filter, the dregs of a decoction and Radix Glycyrrhizae, high mountain horseradish dish, purple bergenia herb, Tabasheer, Lagotis brevituba and Tangut aconite merge, boiling 2 times, the decocting that adds for the first time 10 times of weight boils 2 hours, the decocting that adds for the second time 8 times of weight boils 2 hours, filter, merging filtrate, be concentrated into the clear cream of measuring ρ=1.02～1.04 at 50 ℃, clear cream refrigerates 24 hours to cold-storage jar, get supernatant centrifugal with high speed tube centrifuge, centrifugate continues to be concentrated into the thick paste of measuring ρ=1.07～1.09 at 50 ℃, spraying is dry, must pacify the peaceful extract of youngster,