CN105203469A - Detection method of medicament for treating femoral head necrosis - Google Patents
Detection method of medicament for treating femoral head necrosis Download PDFInfo
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Abstract
The invention discloses a detection method of a medicament for treating femoral head necrosis. The medicament for treating the femoral head necrosis is prepared from the following active pharmaceutical ingredients: pseudo-ginseng, epimedium, salvia miltiorrhiza, drynaria rhizome, flos carthami, achyranthes bidentata blume, teasel root and rhizoma corydalis; the detection method comprises an identification method and a determination method for contents of icariin and salvianolic acid B; the determination method for the contents is easy to operate, high in precision and good in reproducibility; by using a quality control method disclosed by the invention, the quality, in particular to the quality of the medicament for treating the femoral head necrosis, can be accurately and stably controlled so as to adapt to industrial stable production of the medicament.
Description
Technical field
The present invention relates to a kind of Chinese medicine being used for the treatment of caput femoris necrosis, particularly relate to the detection method of this medicine.
Background technology
The symptom of caput femoris necrosis mainly contains hipbone pain, becoming severe at night, joint stuffiness, soreness and weakness of waist and knees.The Chinese herbal granules for the treatment of caput femoris necrosis, for extravasated blood retardance, bone of suffering from a deficiency of the kidney erosion, has promoting blood circulation and removing blood stasis, the merit of the strong bone of kidney tonifying.
The Chinese medicine for the treatment of caput femoris necrosis is that raw material prepares by pseudo-ginseng, barrenwort, the red sage root, the rhizome of davallia, safflower, the root of bidentate achyranthes, teasel root, corydalis tuber.And Chinese medicine preparation steady quality, controlled and have produce repeatability be the premise that medicine has pharmacology and clinical effectiveness repeatability, traditional Chinese medicine quality can be controlled and quality standards in Chinese drugs whether scientific, be the important restriction factor affecting industrialization of Chinese medicine.
Quality controllability is the important indicator of drug assessment, and quality standard is then the imbody of drug quality controllability.So, in order to qualified, steady quality, homogeneous quality caput femoris necrosis medicine can be produced, be necessary to provide a kind of scientific and reasonable Control of drug quality method.
Summary of the invention
The object of this invention is to provide a kind of detection method for the treatment of caput femoris necrosis medicine, to guarantee that the drug quality of producing is stablized controlled.
The detection method for the treatment of caput femoris necrosis medicine provided by the invention is suitable for the medicine be prepared from by following bulk drug: pseudo-ginseng, barrenwort, the red sage root, the rhizome of davallia, safflower, the root of bidentate achyranthes, teasel root, corydalis tuber, described detection method comprises discrimination method and content assaying method.
Wherein, described discrimination method comprises following discriminating:
(1) TLC distinguish of pseudo-ginseng, teasel root: get described medicine 6g, add methyl alcohol 50ml, ultrasonic process 30 minutes, filters, filtrate evaporate to dryness, the residue 50ml that adds water makes dissolving, extract 3 times with ether jolting, each 50ml, discards ether solution, water layer extracts 3 times with water saturated normal butyl alcohol jolting again, each 50ml, merges n-butanol extracting liquid, washs 2 each 50ml with ammonia solution, use the water washing 2 times that normal butyl alcohol is saturated again, each 50ml, divides and gets normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Separately get pseudo-ginseng, each 0.5g of teasel root control medicinal material respectively, be made in the same way of control medicinal material solution; Get Panax Notoginseng saponin R again
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Rb
1reference substance, asperosaponin VI reference substance add methyl alcohol and make every 1ml respectively respectively containing the solution of 1mg, 2.5mg, 2.5mg, 1mg, product solution in contrast; Test according to thin-layered chromatography, draw above-mentioned need testing solution 3 μ l, each 5 μ l of reference substance solution, control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, with the upper solution of normal butyl alcohol-ethyl acetate-butanone-water=2:2:1:4 for developping agent, launch, exhibition is apart from 15cm, take out, dry, spray with 10% ethanol solution of sulfuric acid, be heated to spot development clear;
(2) TLC distinguish of barrenwort: get described medicine 2g, porphyrize, ethyl acetate backflow extracts 1 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes it dissolve, as need testing solution; Separately get barrenwort control medicinal material 0.2g, be made in the same way of control medicinal material solution; Get icariin reference substance 1mg again, add methyl alcohol and dissolve, make 0.5mg/ml reference substance solution; Get barrenwort negative sample 1.2g, then be prepared into negative control solution by need testing solution preparation method; According to thin-layered chromatography test, draw need testing solution, negative solution, the each 5 μ l of reference substance solution, put on same silica gel g thin-layer plate, respectively with methenyl choloride-methanol-water=12:7:3 for developping agent, launch, take out, dry, spray, with 2% aluminum trichloride solution, at 105 DEG C of heating 2min, is inspected under putting 365nm uviol lamp, in test sample chromatogram, on the position corresponding to reference substance, control medicinal material chromatogram, the spot of aobvious same color, and feminine gender is without this spot;
(3) TLC distinguish of tanshin polyphenolic acid B in the red sage root: get this product 1g, add water 50ml, adds hydrochloric acid 0.1ml, ultrasonic process 30 minutes, filter, filtrate extracts 2 times with ethyl acetate jolting, each 30ml, combined ethyl acetate liquid, evaporate to dryness, residue adds absolute ethyl alcohol 1ml makes dissolving, as need testing solution; Separately get red sage root control medicinal material 1g, be made in the same way of control medicinal material solution; Get tanshin polyphenolic acid B reference substance again, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; Test according to thin-layered chromatography, draw each 5 μ l points of above-mentioned solution in same silica G thin layer version, with toluene-methenyl choloride-acetate-methanol-formic acid=2:3:4:0.5:2 for developping agent, launch, exhibition is apart from 15cm, take out, dry, spray with 5% ferric trichloride ethanolic solution, be heated to clear spot, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, reference substance chromatogram, the spot of aobvious same color;
(4) TLC distinguish of the rhizome of davallia: get this product 2g, adds ethyl acetate 30ml, adds hot reflux 1 hour, and filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes it dissolve, as need testing solution; Separately get rhizome of davallia control medicinal material 0.5g, be made in the same way of control medicinal material solution; Separately get aurantiin reference substance appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; According to thin-layered chromatography test, draw need testing solution 5 μ l, control medicinal material solution and each 2 μ L of reference substance solution, put on same silica gel g thin-layer plate, upper liquid is got for developping agent with toluene-ethyl acetate-formic acid-water=1:12:2.5:3, launch, take out, dry, spray is with 2% aluminium choride ethanol test solution, 105 DEG C of heating about 5 minutes, inspect under putting 365nm uviol lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
(5) TLC distinguish of corydalis tuber: get this product 5g, adds strong ammonia solution 3ml, and add diethyl ether 30ml, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Separately get corydalis tuber control medicinal material 1g, be made in the same way of control medicinal material solution; Get tetrahydropalmatine reference substance again, add methyl alcohol and make the solution of every 1ml containing 0.5mg, as need testing solution; According to thin-layered chromatography test, draw each 2 μ L of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane-methenyl choloride-methyl alcohol=15:8:2 for developping agent, launch, take out, dry, put in iodine cylinder and take out after about 3 minutes, after waving the iodine that most plate adsorbs, inspect under putting 365nm uviol lamp, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color;
Described content assaying method comprises the assay method of icariin, content of danshinolic acid B:
The following stated is the assay of icariin:
(1) chromatographic condition: chromatographic column is phenomenexLunaC
18250mm × 4.6mm, 5 μm, mobile phase is acetonitrile-water=25:75, and wavelength is 270nm, and flow velocity is 1.0ml/min, and column temperature is 25 DEG C, and sample size is reference substance solution 5 μ l and need testing solution 10 μ l, and number of theoretical plate calculates should be not less than 4000 by icariin peak;
(2) preparation of reference substance solution: get icariin reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 80 μ g;
(3) preparation of need testing solution: get this product and be about 0.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, weighed weight, add hot reflux 1 hour, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
(4) Determination of Content of Icariin: accurate absorption reference substance solution 5 μ l, need testing solution 10 μ l respectively, injection liquid chromatography, measures by above-mentioned chromatographic condition, with the content of icariin in one point external standard method calculation sample;
The following stated is the assay of tanshin polyphenolic acid B in the red sage root:
(1) chromatographic condition: chromatographic column KromasilC
18250mm × 4.6mm, 5 μm, mobile phase is methanol-acetonitrile-1.7% formic acid water=20:11:69, flow velocity is 1.0ml/min, determined wavelength 286nm, sample size is that reference substance solution, need testing solution respectively enter 10 μ l, and number of theoretical plate calculates should be not less than 6000 by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get tanshin polyphenolic acid B reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 80 μ g, to obtain final product;
(3) preparation of need testing solution: get this product porphyrize, get 0.4g, accurately weighed, put in 50ml measuring bottle, add 75% methyl alcohol and be about 45ml, sonification power 200W, frequency 40kHz, 30 minutes, let cool, add 75% methyl alcohol to scale, shake up, filter, get subsequent filtrate, to obtain final product;
(4) mensuration of content of danshinolic acid B: accurate absorption reference substance solution, each 10 μ l of need testing solution respectively, measure by above-mentioned chromatographic condition, with the content of tanshin polyphenolic acid B in one point external standard method calculation sample.
The present invention establishes the method for quality control of the medicine for the treatment of caput femoris necrosis, in the method set up, the discrimination method mature and feasible of medicinal material, specificity is strong, negative noiseless, content assaying method processing ease, precision is high, favorable reproducibility, use method of quality control of the present invention can accurately, the drug quality of stably Mass Control caput femoris necrosis, to adapt to the industrialization steady production of medicine.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
The TLC distinguish of embodiment 1 pseudo-ginseng, teasel root
Get described medicine 6g, add methyl alcohol 50ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 50ml that adds water makes dissolving, and extract 3 times with ether jolting, each 50ml, discards ether solution; Water layer extracts 3 times with water saturated normal butyl alcohol jolting again, each 50ml, merges n-butanol extracting liquid, washs 2 each 50ml with ammonia solution, use the water washing 2 times that normal butyl alcohol is saturated again, each 50ml, point get normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Separately get pseudo-ginseng, each 0.5g of teasel root control medicinal material respectively, be made in the same way of control medicinal material solution; Get Panax Notoginseng saponin R again
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Rb
1reference substance, asperosaponin VI reference substance add methyl alcohol and make every 1ml respectively respectively containing the solution of 1mg, 2.5mg, 2.5mg, 1mg, product solution in contrast; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw above-mentioned need testing solution 3 μ l, each 5 μ l of reference substance solution, control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, with the upper solution of normal butyl alcohol-ethyl acetate-butanone-water (2:2:1:4) for developping agent, launch, exhibition is apart from 15cm, take out, dry, spray with 10% ethanol solution of sulfuric acid, be heated to spot development clear.Result is respond well, clear spot, durability and favorable reproducibility, negative noiseless.
The TLC distinguish of embodiment 2 barrenwort
Need testing solution preparation method: get described medicine 2g, porphyrize, ethyl acetate backflow extracts 1 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes it dissolve, as need testing solution; Separately get barrenwort control medicinal material 0.2g, be made in the same way of control medicinal material solution; Get icariin reference substance 1mg again, add methyl alcohol and dissolve, make 0.5mg/ml reference substance solution; Get barrenwort negative sample (medicinal material of other the equal recipe quantities beyond removing barrenwort is made) 1.2g, then be prepared into negative control solution by need testing solution preparation method; Test according to thin-layered chromatography, draw need testing solution, negative solution, each 5 μ l of reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-water (12:7:3) for developping agent, launch, take out, dry, spray, with 2% aluminum trichloride solution, at 105 DEG C of heating 2min, is inspected under putting uviol lamp (365nm).In test sample chromatogram, on the position corresponding to reference substance, control medicinal material chromatogram, the spot of aobvious same color, and feminine gender is without this spot.
The TLC distinguish of tanshin polyphenolic acid B in embodiment 3 red sage root
Get this product 1g, add water 50ml, adds hydrochloric acid 0.1ml, ultrasonic process 30 minutes, and filter, filtrate extracts 2 times with ethyl acetate jolting, each 30ml, combined ethyl acetate liquid, evaporate to dryness, and residue adds absolute ethyl alcohol 1ml makes dissolving, as need testing solution; Separately get red sage root control medicinal material 1g, be made in the same way of control medicinal material solution; Get tanshin polyphenolic acid B reference substance again, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 5 μ l points of above-mentioned solution in same silica G thin layer version, with toluene-methenyl choloride-acetate-methanol-formic acid (2:3:4:0.5:2) for developping agent, launch, exhibition, apart from 15cm, is taken out, is dried, spray, with 5% ferric trichloride ethanolic solution, is heated to clear spot; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, reference substance chromatogram, the spot of aobvious same color.Result is respond well, clear spot, durability and favorable reproducibility, negative noiseless.
The TLC distinguish of embodiment 4 rhizome of davallia
Get this product 2g, add ethyl acetate 30ml, add hot reflux 1 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes it dissolve, as need testing solution; Separately get rhizome of davallia control medicinal material 0.5g, be made in the same way of control medicinal material solution; Separately get aurantiin reference substance appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw need testing solution 5 μ l, control medicinal material solution and each 2 μ L of reference substance solution, point is on same silica gel g thin-layer plate, get upper liquid for developping agent with toluene-ethyl acetate-formic acid-water (1:12:2.5:3), launch, take out, dry, spray, with 2% aluminium choride ethanol test solution, 105 DEG C of heating about 5 minutes, is inspected under putting uviol lamp (365nm); In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
The TLC distinguish of embodiment 5 corydalis tuber
Get this product 5g, add strong ammonia solution 3ml, add diethyl ether 30ml, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Separately get corydalis tuber control medicinal material 1g, be made in the same way of control medicinal material solution; Get tetrahydropalmatine reference substance again, add methyl alcohol and make the solution of every 1ml containing 0.5mg, as need testing solution; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 2 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane-methenyl choloride-methyl alcohol (15:8:2) for developping agent, launch, take out, dry, put in iodine cylinder and take out after about 3 minutes, after waving the iodine that most plate adsorbs, inspect under putting uviol lamp (365nm); In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color.
The assay of embodiment 6 icariin
More containing flavour of a drug in prescription of the present invention, its quality control has difficulties, main ingredient in the barrenwort side of being, measures its principal ingredient Icariin content, to this quality of control, ensures that clinical efficacy is significant better.
(1) instrument and reagent
StartoriusCP225D electronic balance (100,000/), Waters2695-2487HPLC, Empower workstation; Millipore water purifior; KQ5200DE type ultrasonic cleaner.Acetonitrile is chromatographically pure; Water is high purity water; It is pure that other chemical reagent is analysis.Icariin reference substance (lot number: 0737-200111, National Institute for Food and Drugs Control).
(2) chromatographic condition
Chromatographic column: phenomenexLunaC
18(250mm × 4.6mm, 5 μm); Mobile phase: acetonitrile-water (25:75); Wavelength: 270nm, flow velocity: 1.0ml/min, column temperature: 25 DEG C, sample size: reference substance solution 5 μ l and need testing solution 10 μ l.Number of theoretical plate calculates should be not less than 4000 by icariin peak.
(3) preparation of reference substance solution
Get icariin reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 80 μ g.
(4) preparation of need testing solution
Get this product and be about 0.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, weighed weight, adds hot reflux 1 hour, lets cool, more weighed weight, supplies the weight of less loss, shake up with methyl alcohol, filters, gets subsequent filtrate, to obtain final product.
(5) Determination of Content of Icariin
Accurate absorption reference substance solution 5 μ l respectively, need testing solution 10 μ l, injection liquid chromatography, measures by above-mentioned chromatographic condition, with the content of barrenwort in one point external standard method calculation sample.
(6) negative sample test
The preparation of barrenwort negative control solution: in prescription flavour of a drug ratio, remove barrenwort, make negative control formulation by preparation technology, then be prepared into negative controls by need testing solution preparation method.Prepare a need testing solution simultaneously, adopt Diode Array Detector peak purity.Accurate absorption need testing solution and each 10 μ l of barrenwort negative control solution, injection liquid chromatography, measures, to obtain final product.Result shows: in barrenwort negative control solution chromatogram, in the retention time (RT=27.5 ~ 30 minute) corresponding to icariin reference substance chromatographic peak, there are no chromatographic peak.Icariin peak purity reaches the requirement of assay, and namely under the condition determination determined, in preparation prescription, the mensuration of other flavour of a drug to icariin is noiseless.
(7) investigation of linear relationship
Precision takes icariin reference substance 8.87mg, puts in 50ml measuring bottle, adds methyl alcohol and dissolves and be diluted to scale, shake up, make storing solution.Accurately from this storing solution respectively draw 2,3,4,6,7, in 8ml to 10ml measuring bottle, respectively with methanol dilution to scale, shake up, obtain 0.03548,0.05322,0.07096,0.10644,0.12418,0.14192mg/ml reference substance solution.The above-mentioned reference substance solution 5 μ l of accurate absorption, injects hplc determination peak area respectively.With reference substance sample size (X) for horizontal ordinate, with peak area (Y) for ordinate, do typical curve, obtaining regression equation is Y=2555X-10.09, r=0.9998.Result shows that icariin sample size is within the scope of 0.151 ~ 0.604mg, and sample size and peak area have good linear relationship, in table 1.
Table 1 icariin linear relationship investigates data
(8) precision test
Accurate absorption need testing solution 10 μ l, injection liquid chromatography, continuous sample introduction 6 times, measurement result is as table 2, and RSD is less than 3%, and illustrate that precision is good, reliability is strong, and method is feasible.
Table 2 precision test
(9) stability test
Accurate absorption, with a test sample solution 10 μ l, respectively at 0,1,3,5,12,24 hour sample introduction, measures peak area, the results are shown in Table 3, and need testing solution was stablized in 24 hours, and RSD is less than 3%.
Table 3 stability test result
(10) replica test
Get same batch sample appropriate, accurately weighed, totally 6 parts, be prepared by need testing solution preparation method respectively, measure peak area, calculate content by one point external standard method, in same lot sample, the mean value of Icariin content is 3.19mg/g, RSD is 1.03%, the results are shown in Table 4, shows that method repeatability is good.
Table 4 replica test result
(11) average recovery test
Take the sample of known content, about 0.25g, totally 6 parts, accurately weighed, the accurate reference substance solution 2ml adding 0.396mg/ml respectively, by need testing solution, preparation method is prepared, sample introduction 10 μ l, measures peak area, calculates the recovery.The results are shown in Table 5, icariin average recovery rate is 98.66%, RSD is 1.91%, illustrates that the content accuracy of the method mensuration icariin is good.
Table 5 recovery test result
The assay of tanshin polyphenolic acid B in embodiment 7 red sage root
(1) instrument and reagent
StartoriusCP225D electronic balance (100,000/), Waters2695-2487HPLC, Empower workstation; Millipore water purifior; KQ5200DE type ultrasonic cleaner.Acetonitrile is chromatographically pure; Water is purified water; It is pure that other chemical reagent is analysis.
Tanshin polyphenolic acid B reference substance (lot number: 0737-200111, by National Institute for Food and Drugs Control).
Strong osseous granules.
(2) chromatographic condition
Chromatographic column KromasilC
18(250mm × 4.6mm, 5 μm); Mobile phase: methanol-acetonitrile-1.7% formic acid water (20:11:69); Flow velocity: 1.0ml/min; Determined wavelength 286nm; Sample size: reference substance solution, need testing solution respectively enter 10 μ l.Number of theoretical plate calculates should be not less than 6000 by tanshin polyphenolic acid B peak.
(3) preparation of reference substance solution
Get tanshin polyphenolic acid B reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 80 μ g, to obtain final product.
(4) preparation of need testing solution
Get this product, porphyrize, get 0.4g, accurately weighed, put in 50ml measuring bottle, add 75% methyl alcohol and be about 45ml, ultrasonic process (power 200W, frequency 40kHz) 30 minutes, lets cool, and adds 75% methyl alcohol to scale, shakes up, and filters, gets subsequent filtrate, to obtain final product.
(5) mensuration of content of danshinolic acid B
Accurate absorption reference substance solution, each 10 μ l of need testing solution, measure by above-mentioned chromatographic condition, with the content of tanshin polyphenolic acid B in one point external standard method calculation sample respectively.
(6) negative sample test
In prescription flavour of a drug ratio, remove the red sage root, make negative control formulation by preparation technology, then be prepared into negative controls by need testing solution preparation method.Prepare a need testing solution simultaneously, adopt Diode Array Detector peak purity.Accurate absorption need testing solution and each 10 μ l of red sage root negative control solution, injection liquid chromatography, measures, to obtain final product.In red sage root negative control solution chromatogram, in the retention time corresponding to tanshin polyphenolic acid B reference substance chromatographic peak there are no chromatographic peak.Namely, under the condition determination determined, in preparation prescription, the mensuration of other flavour of a drug to tanshin polyphenolic acid B is noiseless, and method specificity is strong.
(7) investigation of linear relationship
Precision takes tanshin polyphenolic acid B reference substance 0.01127g, puts in 25ml measuring bottle, adds methyl alcohol and dissolves and be diluted to scale, shake up, make storing solution.Accurately from this storing solution respectively draw 0.5,1,2,3,4, in 5ml to 10ml measuring bottle, respectively with methanol dilution to scale, shake up, obtain a series of reference substance solution.The above-mentioned reference substance solution 10 μ l of accurate absorption, injects hplc determination peak area, the results are shown in Table 6 respectively.With reference substance sample size amount (X) for horizontal ordinate, with peak area (Y) for ordinate, do typical curve, obtaining regression equation is Y=1E+07X-15457(r=0.9999).Result shows, content of danshinolic acid B is within the scope of 0.221 ~ 2.213mg, and content and peak area have good linear relationship.
Table 6 linear relationship investigates result
(8) precision test
Accurate absorption need testing solution 10 μ l, injection liquid chromatography, continuous sample introduction 6 times, measurement result is in table 7.Result shows, the RSD of the chromatographic peak peak area value of tanshin polyphenolic acid B is less than 3%, and illustrate that instrument precision is good, reliability is strong, and method is feasible.
Table 7 precision test
(9) stability test
Accurate absorption, with a test sample solution 10 μ l, respectively at 0,8,12,24,30,36 hour sample introduction, measures peak area, the results are shown in Table 8, and need testing solution is stable in 36h, and RSD value is less than 3%.
Table 8 stability test result
(10) replica test
Get same batch sample appropriate, accurately weighed, totally 6 parts, be prepared by need testing solution preparation method respectively, measure peak area, calculate content by one point external standard method, in same lot sample, the mean value of content of danshinolic acid B is 10.33mg/g, RSD is 2.09%, the results are shown in Table 9, shows that method repeatability is good.
Table 9 replica test result
(11) average recovery test
Take the sample 0.2g of known content, totally 6 parts, accurately weighed, precision adds the reference substance solution 2.15ml that concentration is 0.9624mg/ml respectively, and by need testing solution, preparation method is prepared, sample introduction 10 μ l, measures peak area, calculates the recovery.The results are shown in Table 10, average recovery rate is 98.28%, RSD is 1.50%, and the recovery is good.
Table 10 recovery test result
Claims (1)
1. treat the detection method of caput femoris necrosis medicine for one kind, described treatment caput femoris necrosis medicine is the medicine be prepared from by following bulk drug: pseudo-ginseng, barrenwort, the red sage root, the rhizome of davallia, safflower, the root of bidentate achyranthes, teasel root, corydalis tuber, and described detection method comprises discrimination method and content assaying method;
Wherein, described discrimination method comprises following discriminating:
(1) TLC distinguish of pseudo-ginseng, teasel root: get described medicine 6g, add methyl alcohol 50ml, ultrasonic process 30 minutes, filters, filtrate evaporate to dryness, the residue 50ml that adds water makes dissolving, extract 3 times with ether jolting, each 50ml, discards ether solution, water layer extracts 3 times with water saturated normal butyl alcohol jolting again, each 50ml, merges n-butanol extracting liquid, washs 2 each 50ml with ammonia solution, use the water washing 2 times that normal butyl alcohol is saturated again, each 50ml, divides and gets normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Separately get pseudo-ginseng, each 0.5g of teasel root control medicinal material respectively, be made in the same way of control medicinal material solution; Get Panax Notoginseng saponin R again
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Rb
1reference substance, asperosaponin VI reference substance add methyl alcohol and make every 1ml respectively respectively containing the solution of 1mg, 2.5mg, 2.5mg, 1mg, product solution in contrast; Test according to thin-layered chromatography, draw above-mentioned need testing solution 3 μ l, each 5 μ l of reference substance solution, control medicinal material solution 2 μ l, put respectively on same silica gel g thin-layer plate, with the upper solution of normal butyl alcohol-ethyl acetate-butanone-water=2:2:1:4 for developping agent, launch, exhibition is apart from 15cm, take out, dry, spray with 10% ethanol solution of sulfuric acid, be heated to spot development clear;
(2) TLC distinguish of barrenwort: get described medicine 2g, porphyrize, ethyl acetate backflow extracts 1 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes it dissolve, as need testing solution; Separately get barrenwort control medicinal material 0.2g, be made in the same way of control medicinal material solution; Get icariin reference substance 1mg again, add methyl alcohol and dissolve, make 0.5mg/ml reference substance solution; Get barrenwort negative sample 1.2g, then be prepared into negative control solution by need testing solution preparation method; According to thin-layered chromatography test, draw need testing solution, negative solution, the each 5 μ l of reference substance solution, put on same silica gel g thin-layer plate, respectively with methenyl choloride-methanol-water=12:7:3 for developping agent, launch, take out, dry, spray, with 2% aluminum trichloride solution, at 105 DEG C of heating 2min, is inspected under putting 365nm uviol lamp, in test sample chromatogram, on the position corresponding to reference substance, control medicinal material chromatogram, the spot of aobvious same color, and feminine gender is without this spot;
(3) TLC distinguish of tanshin polyphenolic acid B in the red sage root: get this product 1g, add water 50ml, adds hydrochloric acid 0.1ml, ultrasonic process 30 minutes, filter, filtrate extracts 2 times with ethyl acetate jolting, each 30ml, combined ethyl acetate liquid, evaporate to dryness, residue adds absolute ethyl alcohol 1ml makes dissolving, as need testing solution; Separately get red sage root control medicinal material 1g, be made in the same way of control medicinal material solution; Get tanshin polyphenolic acid B reference substance again, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; Test according to thin-layered chromatography, draw each 5 μ l points of above-mentioned solution in same silica G thin layer version, with toluene-methenyl choloride-acetate-methanol-formic acid=2:3:4:0.5:2 for developping agent, launch, exhibition is apart from 15cm, take out, dry, spray with 5% ferric trichloride ethanolic solution, be heated to clear spot, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, reference substance chromatogram, the spot of aobvious same color;
(4) TLC distinguish of the rhizome of davallia: get this product 2g, adds ethyl acetate 30ml, adds hot reflux 1 hour, and filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes it dissolve, as need testing solution; Separately get rhizome of davallia control medicinal material 0.5g, be made in the same way of control medicinal material solution; Separately get aurantiin reference substance appropriate, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; According to thin-layered chromatography test, draw need testing solution 5 μ l, control medicinal material solution and each 2 μ L of reference substance solution, put on same silica gel g thin-layer plate, upper liquid is got for developping agent with toluene-ethyl acetate-formic acid-water=1:12:2.5:3, launch, take out, dry, spray is with 2% aluminium choride ethanol test solution, 105 DEG C of heating about 5 minutes, inspect under putting 365nm uviol lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
(5) TLC distinguish of corydalis tuber: get this product 5g, adds strong ammonia solution 3ml, and add diethyl ether 30ml, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Separately get corydalis tuber control medicinal material 1g, be made in the same way of control medicinal material solution; Get tetrahydropalmatine reference substance again, add methyl alcohol and make the solution of every 1ml containing 0.5mg, as need testing solution; According to thin-layered chromatography test, draw each 2 μ L of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane-methenyl choloride-methyl alcohol=15:8:2 for developping agent, launch, take out, dry, put in iodine cylinder and take out after about 3 minutes, after waving the iodine that most plate adsorbs, inspect under putting 365nm uviol lamp, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color;
Described content assaying method comprises the assay method of icariin, content of danshinolic acid B:
The following stated is the assay of icariin:
(1) chromatographic condition: chromatographic column is phenomenexLunaC
18250mm × 4.6mm, 5 μm, mobile phase is acetonitrile-water=25:75, and wavelength is 270nm, and flow velocity is 1.0ml/min, and column temperature is 25 DEG C, and sample size is reference substance solution 5 μ l and need testing solution 10 μ l, and number of theoretical plate calculates should be not less than 4000 by icariin peak;
(2) preparation of reference substance solution: get icariin reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 80 μ g;
(3) preparation of need testing solution: get this product and be about 0.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, weighed weight, add hot reflux 1 hour, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
(4) Determination of Content of Icariin: accurate absorption reference substance solution 5 μ l, need testing solution 10 μ l respectively, injection liquid chromatography, measures by above-mentioned chromatographic condition, with the content of icariin in one point external standard method calculation sample;
The following stated is the assay of tanshin polyphenolic acid B in the red sage root:
(1) chromatographic condition: chromatographic column KromasilC
18250mm × 4.6mm, 5 μm, mobile phase is methanol-acetonitrile-1.7% formic acid water=20:11:69, flow velocity is 1.0ml/min, determined wavelength 286nm, sample size is that reference substance solution, need testing solution respectively enter 10 μ l, and number of theoretical plate calculates should be not less than 6000 by tanshin polyphenolic acid B peak;
(2) preparation of reference substance solution: get tanshin polyphenolic acid B reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 80 μ g, to obtain final product;
(3) preparation of need testing solution: get this product porphyrize, get 0.4g, accurately weighed, put in 50ml measuring bottle, add 75% methyl alcohol and be about 45ml, sonification power 200W, frequency 40kHz, 30 minutes, let cool, add 75% methyl alcohol to scale, shake up, filter, get subsequent filtrate, to obtain final product;
(4) mensuration of content of danshinolic acid B: accurate absorption reference substance solution, each 10 μ l of need testing solution respectively, measure by above-mentioned chromatographic condition, with the content of tanshin polyphenolic acid B in one point external standard method calculation sample.
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