CN103454374B - Detection method of bone rehabilitation medicine - Google Patents
Detection method of bone rehabilitation medicine Download PDFInfo
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- CN103454374B CN103454374B CN201310382486.5A CN201310382486A CN103454374B CN 103454374 B CN103454374 B CN 103454374B CN 201310382486 A CN201310382486 A CN 201310382486A CN 103454374 B CN103454374 B CN 103454374B
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Abstract
The invention discloses a quality control method of a bone rehabilitation medicine. The method is characterized in that a method for detecting the content of acacetin-6-C-beta-D-glucoside in oxalis corniculata and a fingerprint spectrum detection method are additionally provided. The method has the advantages that the high performance liquid chromatography is performed for detecting; the operation is simple and convenient; the reproducibility is high; the product quality can be controlled effectively.
Description
Technical field
The present invention relates to the method for quality control of 'Gu Kang ' medicine, belong to the technical field of medicine.
Background technology
Gukang capsule is the kind recorded in Chinese patent drug provincial standard rising national standard part (orthopedics and traumatology fascicle), its quality standard promoted in 2002, main employing thin-layered chromatography carries out Qualitive test to root of Japanese banana medicinal material and pseudo-ginseng, adopts high performance liquid chromatography to carry out quantitative measurement to the psoralen and isopsorapen in Psoralea corylifolia.In order to effectively control product quality further, we have researched and developed a kind of method of creeping oxalis medicinal material being carried out to assay.Creeping oxalis is the main flavour of a drug in Gukang capsule, has clearing heat and promoting diuresis, cool blood detumescence, and effect of the loose stasis of blood of removing toxic substances, for injury from falling down.All can only accomplish Qualitive test all the time, can not accomplish quantitative measurement, in order to effectively control product quality further, we are studied creeping oxalis medicinal material effective ingredient, develop a kind of for controlling oxalic content assaying method.In addition, we have also carried out the research of fingerprint spectrum method to Gukang capsule finished product, and have finally formulated fingerprint atlas detection method.
Summary of the invention
Root of Japanese banana medicinal material is the monarch drug in a prescription of Gukang capsule, but after repeatedly studying, screening, finds to be used for assay without index composition.Creeping oxalis, as the main flavour of a drug in Gukang capsule, has clearing heat and promoting diuresis, cool blood detumescence, and effect of the loose stasis of blood of removing toxic substances, for injury from falling down.Therefore select the index composition in creeping oxalis as another index composition measurement of Gukang capsule.By finding the research of creeping oxalis medicinal material effective ingredient, the effective ingredient in creeping oxalis is robinin-6-C-β-D-Glucose glycosides, therefore have selected robinin-6-C-β-D-Glucose glycosides as another assay index composition in Gukang capsule.
One, robinin-6-C-β in creeping oxalis-D-Glucose glycosides content assaying method research:
1, sample and reagent
Sample: Gukang capsule (lot number: 20100622,20100624,20100701)
Reference substance: robinin-6-C-β-D-Glucose glycosides lot number: 20101107-2 purity: >=98%
Reagent: methyl alcohol, phosphoric acid are pure for analyzing; Methyl alcohol is chromatographically pure
Instrument: Shimadzu AUW-220D type 100,000/electronic balance
Plum Teller AB104-N type ten thousand/electronic balance
Waters 2695-2996 high performance liquid chromatograph
2, liquid-phase condition
Instrument: Waters 2695-2996 high performance liquid chromatograph
Chromatographic column: Diamonsil bis-generation C
18post (250mm × 4.6mm, 5 μm)
Mobile phase: acetonitrile-0.1% phosphoric acid solution (19:81)
Flow velocity: 1.0ml/min
Column temperature: room temperature
3, solution preparation
3.1 need testing solution preparations
Get this product content 1.0g, accurately weighed, put in tool plug conical flask, add methyl alcohol 25ml, weigh, ultrasonic process 30 minutes, lets cool, and supplies the weight of less loss with methyl alcohol, filters, gets subsequent filtrate, to obtain final product;
3.2 reference substance solution preparations
Get robinin-6-C-β-D-Glucose glycosides reference substance 10.48mg, put in 100ml measuring bottle, dissolve with methyl alcohol and be diluted to scale, shaking up, obtain the reference substance stock solution that concentration is 0.1048mg/ml; Precision measures reference substance stock solution 2ml and puts in 100ml measuring bottle again, adds methanol dilution to scale, shakes up, to obtain final product;
3.3 negative sample solution preparations
Get the negative sample of scarce creeping oxalis medicinal material, obtain negative sample solution with test sample preparation method;
The selection of 3.4 determined wavelength
When setting up robinin-6-C-β-D-Glucose glycosides assay to creeping oxalis medicinal material, its uv absorption is scanned, show that robinin-6-C-β-D-Glucose glycosides has absorption maximum at 330nm place, therefore select 330nm to be the wavelength of its assay.See Fig. 1;
3.5 system flexibility researchs
Get robinin-6-C-β-D-Glucose glycosides reference substance solution, need testing solution, negative sample solution measure by above-mentioned liquid-phase condition, the results are shown in Figure 2, Fig. 3, Fig. 4;
Conclusion: Gukang capsule sample chromatogram figure can reach good separation with this understanding, negative sample is noiseless to mensuration;
3.6 different extracting modes are investigated
Take Gukang capsule content 0.5g, six parts, accurately weighed, put in tool plug conical flask, add methyl alcohol 25ml respectively, weigh, investigate backflow 30 minutes, ultrasonic 30 minutes, cold soaking ultrasonic process 30 minutes three kinds of extracting modes after 1 hour, let cool, supply the weight of less loss, filter, get subsequent filtrate, to obtain final product.By above-mentioned form and aspect condition sample introduction, analyze, the results are shown in Table 1;
The different extracting mode of table 1 finished product investigates result
Conclusion: closely, illustrating that backflow, ultrasonic and cold soaking are ultrasonic all can as the disposal route of sample for the result of above 3 kinds of extracting mode gained, but ultrasonic easy and simple to handle, so select the ultrasonic extracting mode as Gukang capsule assay;
3.7 extraction times were investigated
Take Gukang capsule content 0.5g, 6 parts, accurately weighed, put in tool plug conical flask, add methyl alcohol 25ml respectively, weigh, ultrasonic process 20,30,40 minutes, lets cool, supplies the weight of less loss respectively, filters, gets subsequent filtrate, to obtain final product.By above-mentioned form and aspect condition sample introduction, analyze, the results are shown in Table 2;
Table 2 finished product extraction time investigates result
Conclusion: by upper Biao Ke get, within ultrasonic 20,30,40 minutes, its content difference is little, have finally chosen ultrasonic 30 minutes;
3.8 methodological study
3.8.1 linear test
Accurately respectively measure the reference substance solution that concentration is 0.1048mg/ml, with methyl alcohol be made into respectively concentration be 0.05135,0.01712,0.01027,0.01027,0.00642, the reference substance solution of 0.00103mg/ml.Respectively in injection liquid chromatography, record chromatographic peak area, with reference substance concentration for horizontal ordinate, peak area is ordinate mapping, and obtaining regression equation is A=5 × 10
7c+13104, R=0.9996, the results are shown in Table 3 and Fig. 5;
Table 3 linear relationship investigates result
Result shows, robinin-6-C-β-D-Glucose glycosides is between 0.00103 ~ 0.05135mg, and robinin-6-C-β-D-Glucose glycosides amount and peak area are good linear relation;
3.8.2 precision test
Get with a robinin-6-C-β-D-Glucose glycosides reference substance solution, continuous sample introduction six times, calculates its RSD value, the results are shown in Table 4;
Table 4 Precision test result
Result shows, continuous six sample introductions, and average peak area is 2694054, and its RSD value is 1.04%, illustrates that this law precision is good;
3.8.3 replica test
Parallel preparation 6 parts of Gukang capsule finished product (1.2g) need testing solutions measure, and calculate its RSD value, the results are shown in Table 5;
Table 5 replica test result
Result shows, and it is 0.09672 that parallel preparation 6 parts of Gukang capsule need testing solutions measure mean value
mg/g, its RSD value is 1.82%, illustrates that this method repeatability is good;
3.8.4 solution stability testing
Get with a Gukang capsule finished product need testing solution, measure respectively after 0,2,4,8,12,18,24 hour, record peak area, calculates RSD value, the results are shown in Table 6;
Table 6 solution stability testing result
Result shows, stable in 24 hours with a Gukang capsule finished product need testing solution, RSD value is 2.21%, illustrates that this law has good stability;
3.8.5 the recovery
Precision takes the same sample lots content 0.5g of known content, totally 6 parts, add robinin-6-C-β-D-Glucose glycosides reference substance by 80%, 100%, 120% precision respectively, prepare by the preparation method of need testing solution, and sample introduction 10 μ l measures, and the results are shown in Table 7;
Table 7 application of sample recovery test result
Result shows, average recovery rate is 104.69%, and its RSD% value is 0.74%, shows that this law recovery is good;
3.9 assay
By above Method validation, show that the method is linear, precision, repeatability, stability of solution and the recovery all reach requirement, be applicable to the assay of robinin-6-C-β-D-Glucose glycosides in Gukang capsule finished product.We have entered assay to three batches of Gukang capsules, the results are shown in Table 8;
Table 8 Gukang capsule finished product content measurement result
In above 3 batches of Gukang capsule finished products, robinin-6-C-β-D-Glucose glycosides content is about 0.03mg grain, standard can be decided to be robinin-6-C-β-D-Glucose glycosides not lower than 0.02mg/ grain.
Two, finger-print research: on the basis establishing Gukang capsule content assaying method, continue to study the finger-print of Gukang capsule, finally tentatively set up Gukang capsule finger-print system, energy effective control for product quality;
1, sample and reagent
Sample: Gukang capsule (lot number: 20100618,20100619,20100620,20100621,20100623,20100624,20100627,20100701,20100702,20100703)
Reference substance: robinin-6-C-β-D-Glucose glycosides lot number: 20101107-2 purity: 98%
Reagent: methyl alcohol, phosphoric acid are pure for analyzing; Methyl alcohol is chromatographically pure
Instrument: Shimadzu AUW-220D type 100,000/electronic balance
Plum Teller AB104-N type ten thousand/electronic balance
Waters 2695-2996 high performance liquid chromatograph
2, liquid-phase condition
Instrument: Waters 2695-2996 high performance liquid chromatograph
Chromatographic column: Diamonsil bis-generation C
18post (250mm × 4.6mm, 5 μm)
Determined wavelength: 330nm
Flow velocity: 1.0ml/min
Column temperature: room temperature
Mobile phase: acetonitrile is A, 0.1% phosphoric acid solution is B
3, solution preparation
3.1 need testing solution preparations
Get this product content 1.0g, accurately weighed, put in tool plug conical flask, add methyl alcohol 25ml, weigh, ultrasonic process 30 minutes, lets cool, and supplies the weight of less loss with methyl alcohol, filters, gets subsequent filtrate, to obtain final product;
3.2 reference substance solution preparations
Get robinin-6-C-β-D-Glucose glycosides reference substance 10.48mg, put in 100ml measuring bottle, dissolve with methyl alcohol and be diluted to scale, shaking up, obtain the reference substance stock solution that concentration is 0.1048mg/ml; Precision measures reference substance stock solution 2ml and puts in 100ml measuring bottle again, adds methanol dilution to scale, shakes up, to obtain final product;
3.3 measure
Get ten batches of Gukang capsules and make need testing solution by need testing solution preparation method, get in 10 μ l need testing solution injection liquid chromatographies respectively, 10 batches of Gukang capsules measure chromatogram synthesis reference fingerprint and see Fig. 6;
10 batches of Gukang capsule finished product chromatographic datas are imported " similarity evaluation " A version similarity software in 2004, carries out similarity evaluation.Gukang capsule finished product fingerprint similarity result of study table 9;
Table 9 10 batches of Gukang capsule finished product fingerprint similarities
Conclusion: 16 chromatographic peaks indicated in Fig. 6 are as Supplements, and 10 batches of Gukang capsule finished product fingerprint similarities are all more than 0.90.
Accompanying drawing illustrates:
Fig. 1 robinin-6-C-β-D-Glucose glycosides reference substance ultraviolet spectrogram;
Fig. 2 robinin-6-C-β-D-Glucose glycosides reference substance chromatogram;
Fig. 3 Gukang capsule need testing solution chromatogram;
Fig. 4 Gukang capsule lacks creeping oxalis negative sample chromatogram;
Fig. 5 robinin-6-C-β-D-Glucose glycosides linear relationship chart;
Fig. 6 Gukang capsule finished product finger-print.
Embodiment:
Embodiment 1:
Detection method in Gukang capsule is as follows:
1, creeping oxalis assay
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile-0.1% phosphate aqueous solution (19: 81) is mobile phase; Determined wavelength is 330nm.Number of theoretical plate calculates should be not less than 3000 by robinin-6-C-β-D-Glucose glycosides;
The preparation precision of reference substance solution takes robinin-6-C-β-D-Glucose glycosides reference substance in right amount, adds methyl alcohol and makes the solution of every 1ml containing 2 μ g, to obtain final product;
The content under Gukang capsule content uniformity item is got in the preparation of need testing solution, and mixing, gets 1.0g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, weighed weight, ultrasonic process 30 minutes, lets cool, weighed weight again, add the weight that methyl alcohol supplies less loss, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
2, finger-print detects
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent (column length is 25cm, and internal diameter is 4.6mm, and particle diameter is 5 μm); Take acetonitrile as mobile phase A, with 0.1% phosphoric acid solution for Mobile phase B, the regulation according to the form below carries out gradient elution; Flow velocity is 1.0ml/min; Determined wavelength is 330nm.Theoretical cam curve must not be less than 5000 in robinin-6-C-β-D-Glucose glycosides;
The preparation of object of reference solution gets robinin-6-C-β-D-Glucose glycosides reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing robinin-6-C-β-D-Glucose glycosides 0.05mg, to obtain final product;
This product content 1.0g is got in need testing solution preparation, and accurately weighed, put in tool plug conical flask, add methyl alcohol 25ml, weigh, ultrasonic process 30 minutes, lets cool, and supplies the weight of less loss with methyl alcohol, filters, gets subsequent filtrate, to obtain final product;
Determination method precision draws object of reference solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
By similarity evaluation, test sample finger-print and reference fingerprint are through Similarity Measure, and similarity must not lower than 0.90.
Embodiment 2: this method also can be used in bone-recovering tablets, its detection method is as follows:
1, creeping oxalis assay
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile-0.1% phosphate aqueous solution (20: 80) is mobile phase; Determined wavelength is 330nm.Number of theoretical plate calculates should be not less than 3000 by robinin-6-C-β-D-Glucose glycosides;
The preparation precision of reference substance solution takes robinin-6-C-β-D-Glucose glycosides reference substance in right amount, adds methyl alcohol and makes the solution of every 1ml containing 2 μ g, to obtain final product;
The preparation of need testing solution gets 20 bone-recovering tablets, porphyrize, and mixing, gets 1.0g, accurately weighed, puts in tool plug conical flask, precision adds methyl alcohol 25ml, weighed weight, and ultrasonic process 30 minutes, lets cool, more weighed weight, add the weight that methyl alcohol supplies less loss, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
2, finger-print detects
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent (column length is 25cm, and internal diameter is 4.6mm, and particle diameter is 5 μm); Take acetonitrile as mobile phase A, with 0.1% phosphoric acid solution for Mobile phase B, the regulation according to the form below carries out gradient elution; Flow velocity is 1.0ml/min; Determined wavelength is 330nm.Theoretical cam curve must not be less than 5000 in robinin-6-C-β-D-Glucose glycosides;
The preparation of object of reference solution gets robinin-6-C-β-D-Glucose glycosides reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing robinin-6-C-β-D-Glucose glycosides 0.05mg, to obtain final product;
Need testing solution preparation gets 20 bone-recovering tablets, porphyrize, mixing, and get 1.0g, accurately weighed, put in tool plug conical flask, add methyl alcohol 25ml, weigh, ultrasonic process 30 minutes, lets cool, and supplies the weight of less loss with methyl alcohol, filters, gets subsequent filtrate, to obtain final product;
Determination method precision draws object of reference solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
By similarity evaluation, test sample finger-print and reference fingerprint are through Similarity Measure, and similarity must not lower than 0.90.
Embodiment 3: this method also can be used in bone-recovering particles, its detection method is as follows:
1, creeping oxalis assay
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile-0.1% phosphate aqueous solution (21: 79) is mobile phase; Determined wavelength is 330nm.Number of theoretical plate calculates should be not less than 3000 by robinin-6-C-β-D-Glucose glycosides;
The preparation precision of reference substance solution takes robinin-6-C-β-D-Glucose glycosides reference substance in right amount, adds methyl alcohol and makes the solution of every 1ml containing 2 μ g, to obtain final product;
Bone-recovering particles is got in the preparation of need testing solution, porphyrize, and mixing, gets 4.0g, accurately weighed, puts in tool plug conical flask, precision adds methyl alcohol 50ml, weighed weight, and ultrasonic process 30 minutes, lets cool, more weighed weight, add the weight that methyl alcohol supplies less loss, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
2, finger-print detects
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent (column length is 20cm, and internal diameter is 4.6mm, and particle diameter is 5 μm); Take acetonitrile as mobile phase A, with 0.1% phosphoric acid solution for Mobile phase B, the regulation according to the form below carries out gradient elution; Flow velocity is 1.0ml/min; Determined wavelength is 330nm.Theoretical cam curve must not be less than 5000 in robinin-6-C-β-D-Glucose glycosides;
The preparation of object of reference solution gets robinin-6-C-β-D-Glucose glycosides reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing robinin-6-C-β-D-Glucose glycosides 0.05mg, to obtain final product;
Need testing solution preparation gets bone-recovering particles, porphyrize, mixing, and get 1.0g, accurately weighed, put in tool plug conical flask, add methyl alcohol 25ml, weigh, ultrasonic process 30 minutes, lets cool, and supplies the weight of less loss with methyl alcohol, filters, gets subsequent filtrate, to obtain final product;
Determination method precision draws object of reference solution and each 20 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
By similarity evaluation, test sample finger-print and reference fingerprint are through Similarity Measure, and similarity must not lower than 0.90.
Embodiment 4: this method also can be used in bone wound treating medicine wine, and its detection method is as follows:
1, creeping oxalis assay
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile-0.1% phosphate aqueous solution (20: 80) is mobile phase; Determined wavelength is 330nm.Number of theoretical plate calculates should be not less than 3000 by robinin-6-C-β-D-Glucose glycosides;
The preparation precision of reference substance solution takes robinin-6-C-β-D-Glucose glycosides reference substance in right amount, adds methyl alcohol and makes the solution of every 1ml containing 2 μ g, to obtain final product;
The preparation precision of need testing solution measures bone wound treating medicine wine 20ml, puts in 50ml measuring bottle, adds 20ml methyl alcohol, and ultrasonic process 30 minutes, lets cool, and drips methyl alcohol to scale, shakes up, filter, get subsequent filtrate, to obtain final product;
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
2, finger-print detects
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filling agent (column length is 25cm, and internal diameter is 4.6mm, and particle diameter is 5 μm); Take acetonitrile as mobile phase A, with 0.1% phosphoric acid solution for Mobile phase B, the regulation according to the form below carries out gradient elution; Flow velocity is 1.0ml/min; Determined wavelength is 330nm.Theoretical cam curve must not be less than 5000 in robinin-6-C-β-D-Glucose glycosides;
The preparation of object of reference solution gets robinin-6-C-β-D-Glucose glycosides reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing robinin-6-C-β-D-Glucose glycosides 0.05mg, to obtain final product;
Need testing solution is prepared precision and is measured bone wound treating medicine wine 20ml, puts in 50ml measuring bottle, adds 20ml methyl alcohol, and ultrasonic process 30 minutes, lets cool, and drips methyl alcohol to scale, shakes up, filter, get subsequent filtrate, to obtain final product;
Determination method precision draws object of reference solution and each 20 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
By similarity evaluation, test sample finger-print and reference fingerprint are through Similarity Measure, and similarity must not lower than 0.90.
Claims (2)
1. the detection method of 'Gu Kang ' medicine, 'Gu Kang ' medicine is made up of root of Japanese banana, creeping oxalis, teasel root, pseudo-ginseng, Psoralea corylifolia, and its detection method is:
In 'Gu Kang ' medicine, root of Japanese banana discrimination method is: get this product content 5g, add methyl alcohol 30ml, ultrasonic process 30 minutes, and filter, filtrate is concentrated into 1ml, as need testing solution; Separately get root of Japanese banana control medicinal material 1g, be made in the same way of control medicinal material solution; Test according to thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, to take volume ratio as the boiling range of 20:1 be, and the petroleum ether-ethyl acetate of 60 ~ 90 DEG C is developping agent, launches, takes out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear, inspects under putting daylight and 365nm uviol lamp respectively; In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, show spot or the fluorescence spot of same color respectively;
In 'Gu Kang ' medicine, pseudo-ginseng discrimination method is: get this product content 5g, add methyl alcohol 40ml, add hot reflux 1 hour, filter, filtrate evaporate to dryness, the residue 15ml that adds water makes dissolving, extract with methenyl choloride 20ml jolting, discard methenyl choloride liquid, the water saturated normal butyl alcohol jolting of aqueous solution extracts 2 times, first time 20ml, second time 15ml, merges normal butyl alcohol liquid, adds ammonia solution and wash 2 times, first time 30ml, second time 15ml, discards ammonia solution, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Separately get pseudo-ginseng control medicinal material 1g, add methyl alcohol 20ml, be made in the same way of control medicinal material solution; Get notoginsenoside R, ginsenoside Rg1's reference substance again, add methyl alcohol and make every 1ml respectively containing the mixed solution of 2mg, product solution in contrast; Test according to thin-layered chromatography, draw each 2 ~ 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio be 15: 40: 22: 10 methenyl choloride-10 DEG C, acetate-methanol-water with lower floor's solution of left overnight for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material, the spot of aobvious same color;
In 'Gu Kang ' medicine, the content assaying method of Psoralea corylifolia is:
According to high performance liquid chromatography, take octadecylsilane chemically bonded silica as filling agent; Be that the methanol-water of 42: 58 is for mobile phase with volume ratio; Determined wavelength is 245nm; Column temperature 40 DEG C; Number of theoretical plate calculates should be not less than 4500 by psoralen peak, and the degree of separation at psoralen peak and Isopsoralen peak should be greater than 1.5;
The preparation of reference substance solution: get psoralen and isopsorapen reference substance appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1ml containing psoralen 0.25mg, Isopsoralen 0.2mg, to obtain final product;
The preparation of need testing solution: get this product content under content uniformity item, mixing, gets about 0.5g, accurately weighed, put in 25ml measuring bottle, precision adds methyl alcohol 10ml, weighed weight, ultrasonic process 20 minutes, lets cool, weighed weight again, adds the weight that methyl alcohol supplies less loss, shakes up, filter, get subsequent filtrate, filter with miillpore filter, get filtrate, to obtain final product;
Determination method: accurate absorption reference substance solution and each 5 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product;
In 'Gu Kang ' medicine, oxalic content assaying method is:
According to high performance liquid chromatography, be filling agent with octadecylsilane chemically bonded silica; Be that acetonitrile-0.1% phosphate aqueous solution of 15 ~ 25: 85 ~ 75 is for mobile phase with volume ratio; Determined wavelength is 330nm; Number of theoretical plate calculates should be not less than 3000 by robinin-6-C-β-D-Glucose glycosides;
The preparation of reference substance solution: it is appropriate that precision takes robinin-6-C-β-D-Glucose glycosides reference substance, adds methyl alcohol and makes the solution of every 1ml containing 2 μ g, to obtain final product;
The preparation of need testing solution: get the content under 'Gu Kang ' medicine content uniformity item, mixing, gets 1.0g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, weighed weight, ultrasonic process 20 ~ 60 minutes, lets cool, weighed weight again, add the weight that methyl alcohol supplies less loss, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: accurate absorption reference substance solution and each 5 ~ 20 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product;
The fingerprint atlas detection method of 'Gu Kang ' medicine is:
According to high performance liquid chromatography, take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, with 0.1% phosphoric acid solution for Mobile phase B carries out gradient elution, its elution process is: within 0 ~ 10 minute, use 10% mobile phase A and 90% Mobile phase B, within 10 ~ 30 minutes, mobile phase A is gradient to 30% by 10%, Mobile phase B is gradient to 70% by 90%, and within 30 ~ 60 minutes, mobile phase A is gradient to 50% by 30%, and Mobile phase B is gradient to 50% by 70%, within 60 ~ 100 minutes, mobile phase A is gradient to 70% by 50%, and Mobile phase B is gradient to 30% by 50%; Flow velocity is 1.0ml/min; Determined wavelength is 330nm;
Theoretical cam curve must not be less than 5000 in robinin-6-C-β-D-Glucose glycosides;
The preparation of object of reference solution gets robinin-6-C-β-D-Glucose glycosides reference substance in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing robinin-6-C-β-D-Glucose glycosides 0.05mg, to obtain final product;
'Gu Kang ' medicine content 1.0g is got in need testing solution preparation, and accurately weighed, put in tool plug conical flask, add methyl alcohol 25ml, weigh, ultrasonic process 30 minutes, lets cool, and supplies the weight of less loss with methyl alcohol, filters, gets subsequent filtrate, to obtain final product;
Determination method precision draws object of reference solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
2. detection method according to claim 1, is characterized in that this detection method is applicable to bone-recovering tablets agent, granule, pill, injection, soft extract, syrup, mixture, vina, pill, powder.
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| CN107586311B (en) * | 2017-07-25 | 2018-10-09 | 贵州维康子帆药业股份有限公司 | The method that robinin -6-C- β-D-Glucose glycosides is extracted in creeping oxalis |
| CN108375646A (en) * | 2018-02-01 | 2018-08-07 | 贵州维康子帆药业股份有限公司 | A kind of oxalic detection method |
| CN108375645A (en) * | 2018-02-01 | 2018-08-07 | 贵州维康子帆药业股份有限公司 | A kind of detection method of root of Japanese banana |
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| CN100387251C (en) * | 2005-07-28 | 2008-05-14 | 岳顺疆 | Film coated tablet for bone injury and preparation process thereof |
| CN101647942B (en) * | 2009-08-24 | 2012-01-11 | 贵州百灵企业集团制药股份有限公司 | Quality testing method of Yindanxinnaotong soft capsules |
| CN101700306A (en) * | 2009-11-03 | 2010-05-05 | 南京启维医药科技有限公司 | Quality control method of Rupixiao preparation |
| CN102228574A (en) * | 2011-06-30 | 2011-11-02 | 贵阳中医学院 | Preparation method of root of Japanese banana extract and product and application thereof |
| CN102579734B (en) * | 2011-11-28 | 2013-04-24 | 陕西宏府怡悦制药有限公司 | Traditional Chinese medicine composition of bone healing medicine, preparing method thereof and detecting method thereof |
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