CN104034840B - One treats medicine for gynecopathy preparation discrimination method - Google Patents
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Abstract
The invention discloses one and treat medicine for gynecopathy preparation discriminating method of quality control, the technical solution adopted in the present invention adopts TLC distinguish method to differentiate golden cypress, oldenlandia diffusa, rhizoma atractylodis, honey-fried licorice root 4 taste medicine, indentification by TLC golden cypress, rhizoma atractylodis, Radix Glycyrrhizae, oldenlandia diffusa, clear spot, negative control is noiseless is up-to-standard.Adopt the content of Berberine hydrochloride in golden cypress described in high effective liquid chromatography for measuring, Berberine hydrochloride sample size linear relationship between 0.408 ~ 1.836 μ g is good, r=0.9995, average recovery rate is 97.7%, RSD=1.61%, said preparation is in Berberine hydrochloride, and the hydrochloric jamaicin of every 1g particle 1.1 ± 0.165mg is up-to-standard.This method is easy, accuracy good, reproducible, can be used for differentiating that medicament controls the quality of this pharmaceutical preparation.
Description
Technical field
The invention belongs to gynecologic medicine technical field, relate to one and treat medicine for gynecopathy quality of the pharmaceutical preparations discrimination method.
Background technology
In this medicine for gynecopathy preparation use in the past, rely on experience to carry out formula completely to differentiate, by enforcement of the present invention, said preparation can carry out qualitative and fixing quantity, qualitative index can control the formation of compatibility flavour of a drug component substantially, quantitative target provides indicative inspection method, and provides concrete detection embodiment.The present invention, in the quality control application of said preparation, belongs to the technical scheme of carrying out quality control both at home and abroad first.
Summary of the invention
The object of the present invention is to provide one to treat medicine for gynecopathy preparation and differentiate method of quality control, this method is easy, accuracy good, reproducible, can be used for differentiating that medicament controls the quality of this pharmaceutical preparation.
The technical solution adopted in the present invention adopts TLC distinguish method to differentiate golden cypress, oldenlandia diffusa, rhizoma atractylodis, honey-fried licorice root 4 taste medicine, and indentification by TLC golden cypress, rhizoma atractylodis, Radix Glycyrrhizae, oldenlandia diffusa, clear spot, negative control is noiseless is up-to-standard.
Further, adopt the content of Berberine hydrochloride in golden cypress described in high effective liquid chromatography for measuring, Berberine hydrochloride sample size linear relationship between 0.408 ~ 1.836 μ g is good, r=0.9995, average recovery rate is 97.7%, RSD=1.61%, said preparation is in Berberine hydrochloride, and the hydrochloric jamaicin of every 1g particle 1.1 ± 0.165mg is up-to-standard.
Accompanying drawing explanation
Fig. 1 is HPLC chromatogram of the present invention;
Fig. 2 is typical curve of the present invention and linear regression graph.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
The discriminating of golden cypress:
The preparation of need testing solution: get this product 3g, the 20ml that adds water dissolves, and puts evaporate to dryness in water-bath, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution.
The preparation of reference substance solution: get Berberine hydrochloride reference substance, adds methyl alcohol and makes the solution of every 1ml containing lmg, product solution in contrast.
The preparation of negative control solution: remove the medicinal material same treatment beyond golden cypress by prescription, make negative controls.
Thin-layer chromatography: by " Chinese Pharmacopoeia " version one (annex VI B) test in 2010, draw each 5 μ l of above-mentioned 3 kinds of solution respectively, point sample is on same silica gel g thin-layer plate respectively, with normal butyl alcohol one glacial acetic acid one water (7:1:2) for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color, negative fluid is noiseless.
The discriminating of oldenlandia diffusa:
The preparation of need testing solution: get this product 2g, porphyrize, adds 60% ethanol 50mL, cold soaking 1h, ultrasonic process 1h, filter, filtrate adds activated charcoal 0.4g, and 80 DEG C keep 20min, filter, filtrate is concentrated into about 5mL in water-bath, extracts 2 times, each 5mL with sherwood oil (60 ﹣ 90 DEG C) jolting, divide and take off a layer solution, extract 2 times with ethyl acetate jolting, each 5mL, merge ethyl acetate extract, be concentrated into 1mL, as need testing solution.
The preparation of reference substance solution: get oldenlandia diffusa control medicinal material 5g, ultrasonic process 30min after adding ethanol 50mL cold soaking 1h, filter, filtrate adds activated charcoal 0.1g, and 80 DEG C keep 20min, and filter, filtrate is concentrated into about 1mL in water-bath, in contrast medicinal material solution.
The preparation of negative controls: the sample getting scarce oldenlandia diffusa makes negative control solution with need testing solution method.
Thin-layer chromatography: according to thin-layered chromatography (annex VI B) test, draw each 5 μ L of above-mentioned 3 kinds of solution, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, with chloroform-ethyl acetate (4:3) for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control experiments result is noiseless.
The discriminating of rhizoma atractylodis:
The preparation of need testing solution: get this product 3g, adds normal hexane 20ml ultrasonic process 30min, and filter, filtrate volatilizes in water-bath, and residue adds normal hexane 2ml and dissolves as need testing solution.
The preparation of reference substance solution: get rhizoma atractylodis control medicinal material 2g, adds normal hexane 10ml ultrasonic process 30min, and filter, filtrate is evaporate to dryness in water-bath, and residue adds normal hexane 2ml and dissolves product solution in contrast.
The preparation of negative sample solution: the sample getting scarce rhizoma atractylodis makes negative control solution with need testing solution method.
Thin-layer chromatography: according to thin-layered chromatography (annex VI B) test, draw each 5 μ L of above-mentioned 3 kinds of solution, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, with sherwood oil ﹣ ethyl acetate (5: 2) for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control experiments result is noiseless.
The discriminating of honey-fried licorice root:
The preparation of need testing solution: get this product 5g, porphyrize, adds methyl alcohol 40mL, ultrasonic process 30 minutes, filters, filtrate evaporate to dryness, the residue 20mL that adds water makes dissolving, then extracts 2 times with water saturated normal butyl alcohol, each 20ml, merge normal butyl alcohol liquid, wash 1 time with water 20mL, discard water liquid, get normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1mL makes dissolving, as need testing solution.
The preparation of reference substance solution: get control medicinal material 3g, adds the ultrasonic process of methyl alcohol 20ml 30 minutes, filters, filtrate evaporate to dryness, the residue 20mL that adds water makes dissolving, then extracts 2 times with water saturated normal butyl alcohol, each 20ml, merge normal butyl alcohol liquid, wash 1 time with water 20mL, discard water liquid, get normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1mL makes dissolving, product solution in contrast.
The preparation of negative sample solution: the sample getting scarce honey-fried licorice root makes negative sample solution with the preparation method of need testing solution.
Thin-layer chromatography: according to thin-layered chromatography (annex VI B) test, draw each 5 μ L of above-mentioned 3 kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform ﹣ methanol-water (6.6: 3.5:0.5) for developping agent, launch, take out, dry, spray 10% ethanol solution of sulfuric acid, 105 DEG C dry to spot development clear put ultraviolet lamp (365nm) under inspect.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control experiments result is noiseless.
The assay of Berberine hydrochloride:
Chromatographic condition:
Chromatographic column: Agilent (C18,4.6 × 250mm, 5 μm); Determined wavelength: 345nm; Flow velocity: 1.0ml/min; Mobile phase: acetonitrile/triethylamine solution 0.05mol/L (adjusting PH to 3.0 with phosphoric acid) (25:75), sample size: 10 μ L, column temperature is room temperature, and theoretical cam curve calculates by Berberine hydrochloride peak and is not less than 3000.
The preparation of reference substance solution: it is appropriate that precision takes Berberine hydrochloride reference substance, adds methyl alcohol and makes solution about containing 0.102mg in every 1ml, with 0.45 μm of filtering with microporous membrane and get final product.
The preparation of need testing solution: get this product and be about 4g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, weighed weight, ultrasonic process 45 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get the subsequent filtrate of 0.45 μm of filtering with microporous membrane, to obtain final product.
The preparation of negative sample: in ratio and the method for making of prescription, the negative control sample making scarce golden cypress gets 4g, by the preparation method of need testing solution, makes negative control solution.
Specificity is tested:
Get Berberine hydrochloride reference substance solution, need testing solution, negative sample solution injecting chromatograph mensuration, obtain three HPLC chromatograms with the specificity of determination methods.The results are shown in Figure 1.
In Fig. 1, A. Berberine hydrochloride reference substance, A Berberine hydrochloride reference substance, chromatogram transverse axis (X) is retention time, and the longitudinal axis (Y) is for detecting response signal, and baseline stability, at retention time 22.5 ~ 25.0min, occurs response peak; B. heat-clearing is only with particulate samples, and chromatogram transverse axis (X) is retention time, and the longitudinal axis (Y), for detecting response signal, at retention time 22.5 ~ 25.0min, occurs response peak; C. heat-clearing only band particle negative sample, chromatogram transverse axis (X) is retention time, and the longitudinal axis (Y), for detecting response signal, at retention time 22.5 ~ 25.0min, occurs response peak.Fig. 1 shows, sample carries out HPLC mensuration according to the method, and in the retention time identical with reference substance, sample has identical chromatographic peak to occur; Negative control C occurs without identical chromatographic peak in identical retention time, and disturbed specimen does not measure, and has good specificity.
Replica test:
Parallel precision takes same batch this particulate samples 6 parts, and every part is about 0.5g, and precision adds methyl alcohol 5mL, by need testing solution preparation method process, according to above-mentioned chromatographic condition sample introduction 10 μ L, measures and calculates content, the RSD value of peak area.
As shown in table 1 is repeated test determination result (n=6).
Table 1
Precision test:
Draw Berberine hydrochloride reference substance solution, according to the continuous replication 6 of above-mentioned chromatographic condition
Secondary, obtain reference substance peak area, RSD% value.Precision test measurement result (n=6) as shown in table 2.
Table 2
Stability test:
Get test sample by above-mentioned content assaying method, be prepared into need testing solution, get same need testing solution respectively at 0,2,6, within 12,20 hours, measure, to obtain final product. prove that test sample is stable in 20 hours.Stability test measurement result (n=5) as shown in table 3.
Table 3
Linear relationship is investigated:
Precision draws Berberine hydrochloride reference substance solution 4,6,10,12,16,18 μ L with peak area (Y) for ordinate respectively, with sample size (X, μ g) be horizontal ordinate, drawing standard curve, result Berberine hydrochloride is good in 0.408 ~ 1.836 μ g scope internal linear relation, regression equation is y=476944x-365183, r=0.9995.Typical curve as shown in Figure 2 and linear regression graph.
In Fig. 2, transverse axis (X) is sample size, and the longitudinal axis is chromatographic peak area, calculating by returning, showing that Berberine hydrochloride is in 0.408 ~ 1.836 μ g scope, has good linear relationship, this law is used for sample determination, can by content of berberine hydrochloride in linear equation calculation sample.
The typical curve of Berberine hydrochloride shown in table 4 (n=6).
Table 4
Average recovery is tested:
Get known content test sample 6 parts, every part of about 0.25g, accurately weighed, add reference substance solution ml, put in 50ml tool plug conical flask, precision adds methyl alcohol 5ml, weighed weight, ultrasonic process 15 minutes, lets cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, filter, get the subsequent filtrate of 0.45 μm of filtering with microporous membrane, to obtain final product.Application of sample recovery test measurement result (n=6) as shown in table 5.
Table 5
Sample determination:
Get this particulate samples, operate according to above-mentioned need testing solution preparation method and chromatographic condition, sample introduction 10 μ L, measure the content of Berberine hydrochloride.The measurement result (n=10) of Berberine hydrochloride in compound vital energy regualting and blood circulation-promoting particle as shown in table 6.
Table 6
TLC distinguish method is adopted to differentiate 4 taste medicines such as golden cypress, oldenlandia diffusa, rhizoma atractylodis, Radix Glycyrrhizaes; Thin-layer chromatography can differentiate golden cypress, rhizoma atractylodis, Radix Glycyrrhizae, oldenlandia diffusa, and clear spot, negative control is noiseless.The content of Berberine hydrochloride in high effective liquid chromatography for measuring golden cypress, mobile phase: acetonitrile/triethylamine 0.05moL/L solution (adjusting pH to 3.0 with phosphoric acid) (25:75); Determined wavelength: 345nm; Flow velocity: 1.0mL.min-1.Result shows, Berberine hydrochloride sample size linear relationship between 0.408 ~ 1.836 μ g is good, and r=0.9995 (n=6), average recovery rate is 97.7%, RSD=1.61% (n=6).Detect by this method of quality control, said preparation is in Berberine hydrochloride, and the hydrochloric jamaicin 1.1 ± 0.165mg of every 1g particle is criterion of acceptability.
The above is only to better embodiment of the present invention, not any pro forma restriction is done to the present invention, every any simple modification done above embodiment according to technical spirit of the present invention, equivalent variations and modification, all belong in the scope of technical solution of the present invention.
Claims (1)
1. treat a medicine for gynecopathy preparation discrimination method, it is characterized in that: adopt TLC distinguish method to differentiate golden cypress, oldenlandia diffusa, rhizoma atractylodis, honey-fried licorice root 4 taste medicine; Wherein,
The discrimination method of golden cypress is as follows:
1.1) preparation of need testing solution: get preparation 3g, the 20ml that adds water dissolves, and puts evaporate to dryness in water-bath, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution;
1.2) preparation of reference substance solution: get Berberine hydrochloride reference substance, adds methyl alcohol and makes the solution of every 1ml containing lmg, product solution in contrast;
1.3) preparation of negative control solution: remove the medicinal material same treatment beyond golden cypress by prescription, make negative controls;
1.4) thin-layer chromatography: test by " Chinese Pharmacopoeia " version in 2010 annex VI B, draw each 5 μ l of above-mentioned 3 kinds of solution respectively, point sample is on same silica gel g thin-layer plate respectively, with normal butyl alcohol-glacial acetic acid-water 7:1:2 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp 365nm; In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color, negative fluid is noiseless;
The discrimination method of oldenlandia diffusa is as follows:
2.1) preparation of need testing solution: get this product 2g, porphyrize, adds 60% ethanol 50mL, cold soaking 1h, ultrasonic process 1h, filter, filtrate adds activated charcoal 0.4g, and 80 DEG C keep 20min, filter, filtrate is concentrated into about 5mL in water-bath, extracts 2 times, each 5mL with the sherwood oil jolting that boiling range is 60-90 DEG C, divide and take off a layer solution, extract 2 times with ethyl acetate jolting, each 5mL, merge ethyl acetate extract, be concentrated into 1mL, as need testing solution;
2.2) preparation of reference substance solution: get oldenlandia diffusa control medicinal material 5g, ultrasonic process 30min after adding ethanol 50mL cold soaking 1h, filters, filtrate adds activated charcoal 0.1g, and 80 DEG C keep 20min, filter, filtrate is concentrated into about 1mL in water-bath, in contrast medicinal material solution;
2.3) preparation of negative controls: the sample getting scarce oldenlandia diffusa makes negative control solution with need testing solution method;
2.4) thin-layer chromatography: according to thin-layered chromatography annex VI B test, draw each 5 μ L of above-mentioned 3 kinds of solution, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, with chloroform ﹣ ethyl acetate 4:3 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp 365nm; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control experiments result is noiseless;
The discrimination method of rhizoma atractylodis is as follows:
3.1) preparation of need testing solution: get preparation 3g, adds normal hexane 20ml ultrasonic process 30min, and filter, filtrate volatilizes in water-bath, and residue adds normal hexane 2ml and dissolves as need testing solution;
3.2) preparation of reference substance solution: get rhizoma atractylodis control medicinal material 2g, adds normal hexane 10ml ultrasonic process 30min, and filter, filtrate is evaporate to dryness in water-bath, and residue adds normal hexane 2ml and dissolves product solution in contrast;
3.3) preparation of negative sample solution: the sample getting scarce rhizoma atractylodis makes negative control solution with need testing solution method;
3.4) thin-layer chromatography: according to thin-layered chromatography annex VI B test, draw each 5 μ L of above-mentioned 3 kinds of solution, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, with sherwood oil ﹣ ethyl acetate 5:2 for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp 365nm; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control experiments result is noiseless;
The discrimination method of honey-fried licorice root is as follows:
4.1) preparation of need testing solution: get preparation 5g, porphyrize, adds methyl alcohol 40mL, ultrasonic process 30 minutes, filters, filtrate evaporate to dryness, the residue 20mL that adds water makes dissolving, then extracts 2 times with water saturated normal butyl alcohol, each 20ml, merge normal butyl alcohol liquid, wash 1 time with water 20mL, discard water liquid, get normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1mL makes dissolving, as need testing solution;
4.2) preparation of reference substance solution: get control medicinal material 3g, adds the ultrasonic process of methyl alcohol 20ml 30 minutes, filters, filtrate evaporate to dryness, the residue 20mL that adds water makes dissolving, then extracts 2 times with water saturated normal butyl alcohol, each 20ml, merge normal butyl alcohol liquid, wash 1 time with water 20mL, discard water liquid, get normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1mL makes dissolving, product solution in contrast;
4.3) preparation of negative sample solution: the sample getting scarce honey-fried licorice root makes negative sample solution with the preparation method of need testing solution;
4.4) thin-layer chromatography: according to thin-layered chromatography annex VI B test, draw each 5 μ L of above-mentioned 3 kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform ﹣ methanol-water 6.6:3.5:0.5 for developping agent, launch, take out, dry, spray 10% ethanol solution of sulfuric acid, 105 DEG C dry to spot development clear put ultraviolet lamp 365nm under inspect; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color, negative control experiments result is noiseless;
Adopt the content of Berberine hydrochloride in golden cypress described in high effective liquid chromatography for measuring, Berberine hydrochloride sample size linear relationship between 0.408 ~ 1.836 μ g is good, r=0.9995, and average recovery rate is 97.7%, RSD=1.61%; The content assaying method of golden cypress Berberine is specific as follows:
Chromatographic condition:
Chromatographic column: AgilentC18,4.6 × 250mm, 5 μm; Determined wavelength: 345nm; Flow velocity: 1.0ml/min; Mobile phase: acetonitrile/triethylamine solution 0.05mol/L, adjust PH to 3.0,25:75 with phosphoric acid, sample size: 10 μ L, column temperature is room temperature, and theoretical cam curve calculates by Berberine hydrochloride peak and is not less than 3000;
The preparation of reference substance solution: it is appropriate that precision takes Berberine hydrochloride reference substance, adds methyl alcohol and makes solution about containing 0.102mg in every 1ml, with 0.45 μm of filtering with microporous membrane and get final product;
The preparation of need testing solution: get this product and be about 4g, porphyrize, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, weighed weight, ultrasonic process 45 minutes, let cool, weighed weight again, supplies the weight of less loss, shakes up with methyl alcohol, filter, get the subsequent filtrate of 0.45 μm of filtering with microporous membrane and get final product;
The preparation of negative sample: in ratio and the method for making of prescription, the negative control sample making scarce golden cypress gets 4g, by the preparation method of need testing solution, makes negative control solution.
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| US5916919A (en) * | 1997-06-27 | 1999-06-29 | Dalhousie University | Retrovirus protease inhibitors |
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