CN102854282B - Detection method of traditional Chinese medicine compound preparation used for treating laryngopathy - Google Patents
Detection method of traditional Chinese medicine compound preparation used for treating laryngopathy Download PDFInfo
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Abstract
The invention discloses a detection method of a traditional Chinese medicine compound preparation used for treating laryngopathy. The traditional Chinese medicine compound preparation is JinHouSan or granules, buccal tablets, dispersible tablets, effervescent tablets, capsules, or dripping pills prepared with a JinHouSan prescription. According to the invention, qualitative determination is carried out upon an effective component mangiferin in JinHouSan or the granules, buccal tablets, dispersible tablets, effervescent tablets, capsules, or dripping pills prepared with the JinHouSan prescription by using a thin-layer chromatography method; and quantitative determination is carried out upon the effective component mangiferin by using a high-performance liquid chromatography method; or the two methods are adopted simultaneously. With the detection method provided above, the quality of the JinHouSan or the granules, buccal tablets, dispersible tablets, effervescent tablets, capsules, or dripping pills prepared with the JinHouSan prescription can be well controlled, such that stability, homogeneity and controllability of the quality are ensured. Also, the detection method is characterized by convenient operation, good accuracy, high specificity, good stability, and the like.
Description
Technical field
The present invention relates to a kind of detection method of compound Chinese medicinal preparation, particularly relate to a kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease, specifically refer to using golden larynx loose or with the effective constituent mangiferin of the loose little smallflower milkwort herb with root in granule, buccal tablet, dispersing tablet, effervescent tablet, capsule and the dripping pill of making of writing out a prescription of golden larynx as index components, carry out the method for qualitative determination and quantitative measurement.
Background technology
The loose prescription of gold larynx is comprised of Flos Ilicis Asprellae, little smallflower milkwort herb with root, peppermint oil dementholized.Its function with cure mainly as clearing heat and detoxicating, promote the production of body fluid and reduce phlegm, activating blood circulation and reducing swelling pain relieving, is applicable to hoarseness, the larynx symptoms such as vocal cords that acute and chronic pharyngitis and laryngitis cause are hemorrhage, vocal cords oedema, laryngalgia.Little smallflower milkwort herb with root is for forming one of loose prescription of golden larynx, and is the monarch drug in a prescription in prescription, and described monarch drug in a prescription refers to tcm field term, refers to monarch drug in a prescription, ministerial drug, adjutant in traditional Chinese medicine compatibility, makes the monarch drug in a prescription in medicine.Little smallflower milkwort herb with root is as monarch drug in a prescription, the quality of its quality is directly connected to the quality good or not that golden larynx is loose, and current little smallflower milkwort herb with root does not also have effective method of quality control, little smallflower milkwort herb with root quality standard is recorded the local medicinal material standard (version in 1994) in Shanghai, this standard Chinese crude drug differentiates in item to only have proterties to differentiate, do not have thin-layer chromatography to differentiate and assay item, poor, the poor accuracy of this standard specificity as seen, can not control the quality of little smallflower milkwort herb with root effectively.Meanwhile, the 13 the 110th page of the Sanitation Ministry medicine standard Traditional Chinese medicine historical preparation records in the quality standard that golden larynx is loose, without TLC Identification and the content assaying method of effective constituent, can not effectively control the quality that golden larynx is loose.
Summary of the invention
For the deficiencies in the prior art, object of the present invention aims to provide a kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease, the present invention uses thin-layered chromatography, and to the golden larynx for the treatment of larynx disease, the effective constituent mangiferin in granule, buccal tablet, dispersing tablet, effervescent tablet, capsule and dripping pill loose or that make with the loose prescription of golden larynx carries out qualitative determination, use high performance liquid chromatography to carry out quantitative measurement, or use this two kinds of methods simultaneously.By above-mentioned detection method, can control well the quality of this compound Chinese medicinal preparation, guarantee stable, the homogeneous, controlled of its quality.This detection method also has feature easy to operate, that accuracy good, specificity is strong simultaneously.
For achieving the above object, the present invention adopts following technical scheme:
A kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease, described compound Chinese medicinal preparation is golden larynx granule, buccal tablet, dispersing tablet, effervescent tablet, capsule and dripping pill loose or that make with the loose prescription of golden larynx, it is characterized in that, using mangiferin as index components, by thin-layered chromatography, mangiferin is carried out to qualitative determination, carries out according to the following steps:
1) preparation of need testing solution:
A, to get golden larynx loose or appropriate with the loose solid pharmaceutical preparation powder made of writing out a prescription of golden larynx, add extraction solvent, extract the volume mass ratio of addition solid pharmaceutical preparation dust sampling amount loose or that make with the loose prescription of golden larynx with golden larynx of solvent, with ml/g, count 50:1, through ultrasonic processing 15 minutes～90 minutes or heating and refluxing extraction 20 minutes～180 minutes, obtain extracting solution;
B, get the extraction solution that step a obtains, through filtration, filtrate evaporate to dryness, residue, with 10ml methyl alcohol or ethanol, make to dissolve, as need testing solution; Or get the extraction solution that step a obtains, after standing, get supernatant and can be used as need testing solution.
2) preparation of control medicinal material solution: get control medicinal material powder, concrete operations are made control medicinal material solution with step a.
3) preparation of reference substance solution: get mangiferin reference substance, add methyl alcohol and make every 1ml containing the solution of 0.1mg mangiferin, in contrast product solution.
4) differentiate: draw need testing solution, control medicinal material solution 2～10 μ l and reference substance solution 10 μ l, put respectively on same silica gel g thin-layer plate, with developping agent, launch, take out, dry, spray is with developer, in test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, the spot of aobvious same color; Wherein, described developping agent is that volume ratio is ethyl acetate-butanone-formic acid-water of 5～4:3:1:1 or ethyl acetate-formic acid-water that volume ratio is 8～10:1:1, and developer is that concentration is 5% ferric trichloride ethanolic solution.
Preferred: in step 1) in, when described extraction solvent makes concentration be the methyl alcohol of 40%-100% or ethanolic solution that concentration is 40%～100%, get the extraction solution that step a obtains, through filtration, filtrate evaporate to dryness, residue, with 10ml methyl alcohol or ethanol, make to dissolve, as need testing solution.
Preferred: in step 1) in, when described extraction solvent is concentration while being the methyl alcohol of 95%-100% or ethanolic solution that concentration is 95%～100%, get the extraction solution that step a obtains, after standing, get supernatant and can be used as need testing solution.
A kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease, described compound Chinese medicinal preparation is golden larynx granule, buccal tablet, dispersing tablet, effervescent tablet, capsule and dripping pill loose or that make with the loose prescription of golden larynx, it is characterized in that, use high performance liquid chromatography to carry out quantitative measurement to mangiferin, carry out according to the following steps:
1) preparation of need testing solution: get golden larynx solid pharmaceutical preparation powder loose or that make with the loose prescription of golden larynx appropriate, accurately weighed, put in 100ml tool plug conical flask, precision adds extraction solvent, extract the volume mass ratio of addition solid pharmaceutical preparation dust sampling amount loose or that make with the loose prescription of golden larynx with golden larynx of solvent, in ml/g 200:1, close plug, weighed weight, ultrasonic processing 25～90 minutes or refluxing extraction 0.5～3 hour, let cool, weighed weight, with extracting accordingly the weight that solvent is supplied less loss, shakes up again, filter, get subsequent filtrate as need testing solution;
2) preparation of reference substance solution: get mangiferin reference substance appropriate, accurately weighed, add concentration and be 60% ethanol and be prepared into the reference substance solution that concentration is 20.584～1029.2 μ g/ml, obtain;
3) chromatographic condition and system suitability: acetonitrile-methyl alcohol-aqueous acid that methyl alcohol-aqueous acid that acetonitrile-aqueous acid that the volume ratio of take is 10～20:90～80 or volume ratio are 10～40:90～60 or volume ratio are 0～10:10～30:90～60 is liquid mobile phase; The detection wavelength of high performance liquid chromatography is that 240～365nm, column temperature are that 25～35 ℃, chromatographic column are C18 post, and theoretical cam curve is calculated and should be not less than 8000 by mangiferin peak;
4) measure: precision is drawn reference substance solution and need testing solution 5 μ l respectively, inject high performance liquid chromatograph, measure, obtain.
Preferred: in step 1), described extraction solvent is that concentration is the ethanol that 50～70% methyl alcohol or concentration are 50～70%.
Preferred: in step 3), the acid in described aqueous acid is selected from phosphoric acid, acetic acid, formic acid, the acid in described aqueous acid and the volume ratio of water are 0.1～0.5:100.
A kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease, described compound Chinese medicinal preparation is golden larynx granule, buccal tablet, dispersing tablet, effervescent tablet, capsule and dripping pill loose or that make with the loose prescription of golden larynx, it is characterized in that, using mangiferin as index components, use thin-layered chromatography to carry out qualitative determination to mangiferin, meanwhile, use high performance liquid chromatography to carry out quantitative measurement to mangiferin in little smallflower milkwort herb with root; Their concrete operation step as mentioned before.
Beneficial effect of the present invention is:
Little smallflower milkwort herb with root is as monarch drug in a prescription, and the quality of its quality is directly connected to the quality good or not that golden larynx is loose, because current little smallflower milkwort herb with root does not have effective method of quality control, so it is carried out to quality standard research.In research process, with thin layer identification color spectral technology and high-efficient liquid phase chromatogram technology, the chemical composition of little smallflower milkwort herb with root is analyzed, found mangiferin composition, this mangiferin composition is used purchased from National Institute for Food and Drugs Control (lot number 111607-200402) mangiferin reference substance and is compared, and its chromatogram feature is consistent.As: with the developping agent of multiple opposed polarity, carry out thin layer discriminating, little smallflower milkwort herb with root test sample with the corresponding position of mangiferin reference substance on the feature spot of aobvious same color; With high performance liquid chromatograph (diode array detector DAD), measure, little smallflower milkwort herb with root test sample has identical abosrption spectrogram with mangiferin reference substance; With the mobile phase of multiple volume ratio, measure, little smallflower milkwort herb with root test sample is having identical absorption peak with on the corresponding position of reference substance again, illustrates and in little smallflower milkwort herb with root, contains mangiferin composition.In little smallflower milkwort herb with root, having mangiferin composition is that inventor finds first.Mangiferin has the central nervous system of inhibition, anti-inflammatory, antibacterial, anti-herpes simplex virus; cholagogic and immunization; to simulated high altitude acute hypoxia, hepatic injury has protective effect; pharmacological evaluation shows; this product is used for the treatment of chronic bronchitis good therapeutic effect; visible mangiferin is effective constituent in little smallflower milkwort herb with root, and the quality that the mangiferin of usining is controlled little smallflower milkwort herb with root as main evaluation index has realistic meaning.Compared with prior art, the present invention has the following advantages: the present invention is usingd mangiferin as index components, use thin-layered chromatography to carry out qualitative determination to mangiferin, or use high performance liquid chromatography to carry out quantitative measurement to mangiferin in little smallflower milkwort herb with root, also can use these two kinds of methods simultaneously, method of the present invention can be controlled golden larynx other formulation loose and that make with the loose prescription of golden larynx well, described other formulation comprises granule, buccal tablet, dispersing tablet, effervescent tablet, capsule and dripping pill, guarantee stable, the homogeneous of its quality, controlled.The advantages such as method of the present invention is easy to operate, accuracy good, specificity is strong, good stability.
Accompanying drawing explanation
Fig. 1 is thin layer identification color spectrogram of the present invention, in figure: 1 is the loose test sample of golden larynx, and 2 negative samples, 3 is little smallflower milkwort herb with root control medicinal material, 4 is mangiferin reference substance.
Fig. 2 is assay reference substance high-efficient liquid phase chromatogram.
Fig. 3 is assay test sample high-efficient liquid phase chromatogram.
The negative sample high-efficient liquid phase chromatogram of Fig. 4.
Embodiment
In detection method provided by the invention, agents useful for same is buied by market, the reagent of wherein differentiating for thin-layer chromatography is pure for analyzing, the methyl alcohol and the ethanol that for high efficiency liquid phase, carry out assay are chromatographically pure, gold larynx falls apart and other formulation is made by Community in Baiyunshan, Guangzhou Jing Xiutang medicine company incorporated company, and reference substance mangiferin is purchased from National Institute for Food and Drugs Control (lot number 111607-200402).
Be below TLC Identification embodiment of the present invention, described embodiment is in order to further describe the present invention, rather than restriction the present invention.
One, thin-layer chromatography is differentiated:
Embodiment 1:
The preparation of need testing solution: get golden larynx divided powder 0.5g, add concentration and be 40% methyl alcohol 25ml, ultrasonic processing 15 minutes, filtration, filtrate evaporate to dryness, residue make to dissolve with the methyl alcohol of 10ml, obtain.
The preparation of control medicinal material solution: get control medicinal material powder, prepare control medicinal material solution by the preparation method of need testing solution.
The preparation of reference substance solution: get mangiferin reference substance, add methyl alcohol and make every 1ml containing the reference substance solution of 0.1mg.
Differentiate: draw need testing solution, control medicinal material solution 2 μ l and reference substance solution 10 μ l, put respectively on same silica gel g thin-layer plate, ethyl acetate-butanone-formic acid-the water (volume ratio is 5:3:1:1) of take is developping agent, launch, take out, dry, the ferric trichloride ethanolic solution that concentration is 5% is take in spray, in test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, the spot of aobvious same color.
Thin-layer chromatography differentiates that durability investigates: investigated respectively under cryogenic conditions under (4 ℃), room temperature condition under (25 ℃), low humidity condition the separating effect of (humidity is 75%) principal spot under (humidity is 30%), super-humid conditions.Test findings shows principal spot good separating effect, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, equal spots of aobvious same color.This discrimination method stability, reproducible is described.
Specificity is examined wiping: by the 13 the 110th page of the Sanitation Ministry medicine standard Traditional Chinese medicine historical preparation, record [prescription] in the quality standard that golden larynx is loose and remove little smallflower milkwort herb with root medicinal material, by [method for making], prepare negative sample, get negative sample powder and prepare negative sample solution by the preparation method of aforementioned need testing solution, by the preparation method of aforementioned control medicinal material solution, prepare control medicinal material solution, preparation method by aforementioned reference substance solution prepares reference substance solution, by aforementioned discrimination method, differentiate, test findings shows that negative sample solution does not disturb measuring, illustrate that specificity is good.See accompanying drawing 1.
Embodiment 2: concrete operation step is with embodiment 1, and different is in the preparation process of need testing solution: ultrasonic processing 30 minutes; In differentiating step: the ethyl acetate-butanone-formic acid-water (volume ratio is 4:3:1:1) of take is developping agent.
Embodiment 3: concrete operation step is with embodiment 1, and different is in the preparation process of need testing solution: ultrasonic processing 90 minutes;
Embodiment 4: concrete operation step is with embodiment 1, and different is in the preparation process of need testing solution: extracting solvent is that concentration is 50% methyl alcohol, adds hot reflux 20 minutes; Differentiate in step: the ethyl acetate-formic acid-water (volume ratio is 8:1:1) of take is developping agent.
Embodiment 5: concrete operation step is with embodiment 1, and different is in the preparation process of need testing solution: extracting solvent is 40% ethanol, adds hot reflux 60 minutes, and residue makes to dissolve with 10ml ethanol; Differentiate in step: the ethyl acetate-formic acid-water (volume ratio is 9:1:1) of take is developping agent.
Embodiment 6: concrete operation step is with embodiment 1, and different is in the preparation process of need testing solution: extract solvent and add 50% ethanol, add hot reflux 180 minutes, differentiate in step: the ethyl acetate-formic acid-water (volume ratio is 10:1:1) of take is developping agent.
Embodiment 7: concrete operation step is with embodiment 1, different is in the preparation process of need testing solution: extracting solvent is that concentration is 95% methyl alcohol, extract obtains (extract does not need to filter and evaporate to dryness) after standing, differentiates in step: the point sample amount of need testing solution, control medicinal material solution is 10 μ l.
Embodiment 8: concrete operation step is with embodiment 1, different is in the preparation process of need testing solution: the methyl alcohol that extraction solvent strength is 100%, extract obtains (extract does not need to filter and evaporate to dryness) after standing, differentiates in step: the point sample amount of need testing solution, control medicinal material solution is 5 μ l.
Embodiment 9: concrete operation step is with embodiment 1, and different is in the preparation process of need testing solution: extracting solvent is that concentration is 95% ethanol, and filtrate filters and get final product, and differentiate in step: the point sample amount of need testing solution, control medicinal material solution is 10 μ l.
Embodiment 10: concrete operation step is with embodiment 1, and different is in the preparation process of need testing solution: extracting solvent is that concentration is 100% ethanol, and filtrate filters and get final product, and differentiate in step: the point sample amount of need testing solution, control medicinal material solution is 10 μ l.
Embodiment 11: concrete operation step is with embodiment 1, and different is in the preparation process of need testing solution: sample is for making buccal tablet, dispersing tablet, effervescent tablet, capsule or the dripping pill sample after pulverizing and cross 65 sieves with the loose prescription of golden larynx.
Two, mangiferin assay:
Embodiment 12:
The preparation of need testing solution: get the about 0.25g of golden larynx divided powder, accurately weighed, put in 100ml tool plug conical flask, it is 50% methyl alcohol 50ml that precision adds concentration, close plug, weighed weight, ultrasonic processing 25 minutes, let cool, weighed weight again, supplies the weight of less loss with the methyl alcohol that concentration is 50%, shake up, filter, get subsequent filtrate as need testing solution.
The preparation of reference substance solution: get mangiferin reference substance appropriate, accurately weighed, add concentration and be 60% ethanol and be prepared into the reference substance solution that concentration is 400 μ g/ml, obtain.
Chromatographic condition and system suitability: the acetonitrile-0.1% phosphorus aqueous acid that the volume ratio of take is 15:85 is mobile phase; The detection wavelength of high performance liquid chromatography is that 258nm, column temperature are that 25 ℃, chromatographic column are C18 post, and theoretical cam curve is calculated and should be not less than 8000 by mangiferin peak;
Measure: precision is drawn reference substance solution and need testing solution 5 μ l respectively, inject high performance liquid chromatograph, measure, obtain.
Embodiment 13: concrete operation step is with embodiment 12, and different is in the preparation process of need testing solution: with 50% ethanol, be to extract solvent, adopt ultrasonic processing 45 minutes; In chromatographic condition and system suitability step: the acetonitrile-0.2% phosphorus aqueous acid that the volume ratio of take is 10:90 is mobile phase; The detection wavelength of high performance liquid chromatography is 240nm.
Embodiment 14: concrete operation step is with embodiment 12, and different is in the preparation process of need testing solution: with 70% ethanol, be to extract solvent, adopt ultrasonic processing 90 minutes; In chromatographic condition and system suitability step: the acetonitrile-0.5% phosphorus aqueous acid that the volume ratio of take is 20:80 is mobile phase; The detection wavelength of high performance liquid chromatography is 365nm.
Embodiment 15: concrete operation step is with embodiment 12, and different is in the preparation process of need testing solution: with 50% methyl alcohol, be to extract solvent, adopt refluxing extraction 0.5 hour; In chromatographic condition and system suitability step: the methyl alcohol-0.1% phosphorus aqueous acid that the volume ratio of take is 10:90 is mobile phase.
Embodiment 16: concrete operation step is with embodiment 12, and different is in the preparation process of need testing solution: with 60% methyl alcohol, be to extract solvent, adopt refluxing extraction 1 hour; In chromatographic condition and system suitability step: the methyl alcohol-0.1% phosphorus aqueous acid that the volume ratio of take is 10:90 is mobile phase.
Embodiment 17: concrete operation step is with embodiment 12, and different is in the preparation process of need testing solution: with 70% methyl alcohol, be to extract solvent, adopt refluxing extraction 1 hour; In chromatographic condition and system suitability step: the methyl alcohol-0.1% vinegar aqueous acid that the volume ratio of take is 20:80 is mobile phase.
Embodiment 18: concrete operation step is with embodiment 12, and different is in the preparation process of need testing solution: adopt refluxing extraction 3 hours; In chromatographic condition and system suitability step: the methyl alcohol-0.2% vinegar aqueous acid that the volume ratio of take is 40:60 is mobile phase.
Embodiment 19: concrete operation step is with embodiment 12, and different is in chromatographic condition and system suitability step: the methyl alcohol-0.5% vinegar aqueous acid that the volume ratio of take is 40:60 is mobile phase, and column temperature is 30 ℃.
Embodiment 20: concrete operation step is with embodiment 11, and different is in chromatographic condition and system suitability step: the acetonitrile-methyl alcohol-0.1% phosphorus aqueous acid that the volume ratio of take is 0:10:90 is mobile phase, and column temperature is 35 ℃.
Embodiment 21: concrete operation step is with embodiment 12, and different is in chromatographic condition and system suitability step: the acetonitrile-methyl alcohol-0.1% first aqueous acid that the volume ratio of take is 5:15:80 is mobile phase.
Embodiment 22: concrete operation step is with embodiment 12, and different is in chromatographic condition and system suitability step: the acetonitrile-methyl alcohol-0.2% first aqueous acid that the volume ratio of take is 10:30:60 is mobile phase.
Embodiment 23: concrete operation step is with embodiment 12, and different is in chromatographic condition and system suitability step: the acetonitrile-methyl alcohol-0.5% first aqueous acid that the volume ratio of take is 10:30:60 is mobile phase.
Embodiment 24: concrete operation step is with embodiment 12, and different is in the preparation process of need testing solution: sample is for making buccal tablet, dispersing tablet, effervescent tablet, capsule or the dripping pill sample after pulverizing and cross 65 sieves with the loose prescription of golden larynx.
Content assaying method is learned checking:
Instrument and reagent:
High performance liquid chromatograph: Agilent 1260LC; DAD detecting device.Ultrasound Instrument: the KQ-600DE of Kunshan Ultrasonic Instruments Co., Ltd. type.
Reference substance mangiferin is purchased from National Institute for Food and Drugs Control (lot number 111607-200402), and golden larynx is loose to be made by Community in Baiyunshan, Guangzhou Jing Xiutang medicine company incorporated company; (chromatographically pure, honeywell), the ethanol of using as solvent and methyl alcohol are analytical reagent to acetonitrile, are purchased from Guangzhou chemical reagent work.
Chromatographic condition and system suitability: the acetonitrile-0.1% phosphorus aqueous acid that the volume ratio of take is 15:85 is mobile phase; The detection wavelength of high performance liquid chromatography is that 258nm, column temperature are 25 ℃, chromatographic column: phenomenex synergi FUsion-RP (4 μ m, 4.6 * 250mm), theoretical cam curve is calculated and should be not less than 8000 by mangiferin peak.
The preparation of need testing solution: get the about 0.25g of golden larynx divided powder, accurately weighed, put in 100ml tool plug conical flask, it is 60% ethanol 50ml that precision adds concentration, close plug, weighed weight, ultrasonic processing 30 minutes, let cool, weighed weight again, supplies the weight of less loss with the ethanol that concentration is 60%, shake up, filter, get subsequent filtrate as need testing solution; Its high-efficient liquid phase chromatogram as shown in Figure 3.
The preparation of reference substance solution: get mangiferin reference substance appropriate, accurately weighed, add concentration and be 60% ethanol and be prepared into the reference substance solution that concentration is 400 μ g/ml, obtain.Its high-efficient liquid phase chromatogram as shown in Figure 2.
Linear relationship is investigated: precision takes mangiferin reference substance 0.05146g, put in 50ml volumetric flask, adding concentration and be 60% ethanol dissolves and is settled to scale, measure respectively 2ml, 2ml, 2ml, 2ml, 5ml puts in the volumetric flask of 100ml, 50ml, 25ml, 10ml, 10ml, add concentration and be 60% ethanol and be settled to scale, being mixed with serial content is the mangiferin reference substance solution of 20.584～1029.2 μ g/ml.Accurate each the 5 μ l of above-mentioned reference substance solution that draw, under aforementioned chromatographic condition, measure, and with peak area Y be ordinate, concentration X(n=6) for horizontal ordinate carries out linear regression, obtaining regression equation is Y=21.207X-10.068, r=0.9999, mangiferin is good linear relationship at 20.584～1029.2 μ g/ml.Test findings is in Table 1.
Table 1 linear relationship is investigated data
| Tested number | Concentration (μ g/ml) | Peak area |
| 1 | 20.584 | 434.2 |
| 2 | 41.168 | 860.9 |
| 3 | 82.336 | 1729.9 |
| 4 | 205.84 | 4330.05 |
| 5 | 514.6 | 10943.85 |
| 6 | 1029.2 | 21801.8 |
Precision test: the accurate mangiferin reference substance solution 5 μ l that draw, repeat sample introduction 6 times, measure peak area, the RSD that calculates mangiferin peak area is 0.08%.Result shows that instrument precision is good.The results are shown in Table 2.
Table 2 precision test data
Replica test: by 6 parts of need testing solutions of the parallel preparation of preparation method of aforementioned need testing solution, measure peak area, according to the content of regression equation calculation mangiferin.The content mean value of result mangiferin is that RSD is 0.24%, shows that repeatability is good.The results are shown in Table 3.
Table 3 replica test data
Stability test: precision was drawn need testing solution 5 μ l at 0,4,8,12,16,22 hour, sample introduction analysis respectively, the peak area of mensuration mangiferin, RSD is 0.26%.Show in test sample 22 hours stable.The results are shown in Table 4.
Table 4 stability test data
Application of sample recovery test: get 6 parts of the test samples of known content, every part of 0.15g, accurately weighed, put in 100ml conical flask, to add 5ml concentration be the reference substance solution of 1029.2 μ g/ml to precision respectively, according to aforementioned need testing solution preparation method, prepares and measure.The average recovery of this method mangiferin is between 98%～100.2%, and RSD is 1.04%.The results are shown in Table 5.
Table 5 application of sample recovery test data
Different manufacturers chromatographic column is investigated: get the loose sample of golden larynx, by the preparation method of aforementioned need testing solution, prepare test sample, by the chromatographic column of different brands, carry out assay.Chromatographic column 1:Phenemenex Synergi 4u Fusion-RP 80A 4.6 * 250mm NO:388211-23, chromatographic column 2:Agilent Eclipse Plus C18 5um 4.6 * 250mm SN:USUXA01394, chromatographic column 3:Agilent TC-C18 5um 4.6 * 250mm SN:USEGK01101.Test findings shows that 3 root chromatogram columns of investigating are all applicable to this method.The results are shown in 6.
The different chromatographic columns of table 6 are investigated data
Specificity is investigated: by the 13 the 110th page of the Sanitation Ministry medicine standard Traditional Chinese medicine historical preparation, record [prescription] in the quality standard that golden larynx is loose and remove little smallflower milkwort herb with root medicinal material, by [method for making], prepare negative sample, get negative sample powder and prepare negative sample solution by the preparation method of aforementioned need testing solution, by aforementioned chromatographic condition and system suitability condition, measure, test findings shows that negative sample solution does not disturb measuring.See accompanying drawing 4.
More than test shows that the method specificity of this assay is good, and precision is high, and stability, repeatability, accuracy are good.
For a person skilled in the art, can make other various corresponding changes and distortion according to technical scheme described above and design, and these all changes and distortion all should belong to the protection domain of the claims in the present invention within.
Claims (4)
1.
a kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease, described compound Chinese medicinal preparation is golden larynx granule, buccal tablet, dispersing tablet, effervescent tablet, capsule and dripping pill loose or that make with the loose prescription of golden larynx, it is characterized in that, using mangiferin as index components, by thin-layered chromatography, mangiferin is carried out to qualitative determination, carries out according to the following steps:
1) preparation of need testing solution:
a, to get golden larynx loose or appropriate with the loose solid pharmaceutical preparation powder made of writing out a prescription of golden larynx, add extraction solvent, extract the volume mass ratio of addition solid pharmaceutical preparation dust sampling amount loose or that make with the loose prescription of golden larynx with golden larynx of solvent, with ml/g, count 50:1, through ultrasonic processing 15 minutes～90 minutes or heating and refluxing extraction 20 minutes～180 minutes, obtain extracting solution;
b, get the extraction solution that step a obtains, through filtration, filtrate evaporate to dryness, residue, with methyl alcohol or ethanol, make to dissolve, as need testing solution; Or get the extraction solution that step a obtains, after standing, get supernatant and can be used as need testing solution;
in step 1) in, described extraction solvent is that concentration is the methanol solution of 95%-100%, gets the extraction solution that step a obtains, and after standing, get supernatant and can be used as need testing solution;
2) preparation of control medicinal material solution: get control medicinal material powder, concrete operations are made control medicinal material solution with step a;
3) preparation of reference substance solution: get mangiferin reference substance, add methyl alcohol and make every 1ml containing the solution of 0.1mg mangiferin, in contrast product solution;
4) differentiate: draw need testing solution, control medicinal material solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with developping agent, launch, take out, dry, spray is with developer, in test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, the spot of aobvious same color; Wherein, described developping agent is that volume ratio is ethyl acetate-butanone-formic acid-water of 5～4:3:1:1 or ethyl acetate-formic acid-water that volume ratio is 8～10:1:1, and developer is that concentration is 5% ferric trichloride ethanolic solution.
2.
a kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease as claimed in claim 1, is characterized in that, uses high performance liquid chromatography to carry out quantitative measurement to mangiferin, carries out according to the following steps:
1) preparation of need testing solution: get golden larynx solid pharmaceutical preparation powder loose or that make with the loose prescription of golden larynx appropriate, accurately weighed, put in tool plug conical flask, precision adds extraction solvent, extract the volume mass ratio of addition solid pharmaceutical preparation dust sampling amount loose or that make with the loose prescription of golden larynx with golden larynx of solvent, in ml/g 200:1, close plug, weighed weight, ultrasonic processing 25～90 minutes or refluxing extraction 0.5～3 hour, let cool, weighed weight, with extracting accordingly the weight that solvent is supplied less loss, shakes up again, filter, get subsequent filtrate as need testing solution;
2) preparation of reference substance solution: get mangiferin reference substance appropriate, accurately weighed, add concentration and be 60% ethanol and be prepared into the reference substance solution that concentration is 20.584～1029.2 μ g/ml, obtain;
3) chromatographic condition and system suitability: acetonitrile-methyl alcohol-aqueous acid that methyl alcohol-aqueous acid that the volume ratio of take is 10～40:90～60 or volume ratio are 0～10:10～30:90～60 is liquid mobile phase; The detection wavelength of high performance liquid chromatography is that 240～365nm, column temperature are that 25～35 ℃, chromatographic column are C18 post, and theoretical cam curve is calculated and should be not less than 8000 by mangiferin peak;
4) measure: precision is drawn reference substance solution and need testing solution respectively, inject high performance liquid chromatograph, measure, obtain.
3.
a kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease as claimed in claim 2, is characterized in that: in step 1), described extraction solvent is that concentration is 50～70% methyl alcohol.
4.
a kind of detection method for the treatment of the compound Chinese medicinal preparation of larynx disease as claimed in claim 2, it is characterized in that: in step 3), acid in described aqueous acid is selected from phosphoric acid, acetic acid, formic acid, and the acid in described aqueous acid and the volume ratio of water are 0.1～0.5:100.
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