CN104820055B - A kind of discrimination method of attached sweet medicine - Google Patents
A kind of discrimination method of attached sweet medicine Download PDFInfo
- Publication number
- CN104820055B CN104820055B CN201510241416.7A CN201510241416A CN104820055B CN 104820055 B CN104820055 B CN 104820055B CN 201510241416 A CN201510241416 A CN 201510241416A CN 104820055 B CN104820055 B CN 104820055B
- Authority
- CN
- China
- Prior art keywords
- solution
- reference substance
- aconine
- benzoyl
- extraction column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The discrimination method of a kind of attached sweet pharmaceutical preparation of the present invention, differentiates Radix Aconiti Lateralis Preparata by high performance liquid chromatography, and its chromatographic column connects phenyl bonded silica for filler with polarity ether, Radix Glycyrrhizae is differentiated by thin layer chromatography, can be easy to be accurate, specificity is strong, the attached sweet medicine of discriminating that ruggedness is good.
Description
Technical field
The present invention relates to medicine, be specifically related to the discrimination method of a kind of attached sweet medicine, belong to field of pharmaceutical technology.
Technical background
A kind of medicine treating allergic rhinitis and bronchial asthma of Chinese invention patent (ZL201210536161.3), by Radix Aconiti Lateralis Preparata 6-18 part, Radix Glycyrrhizae 1-9 part is that raw material makes (being called for short " attached sweet medicine "), has good curative effect for allergic rhinitis and bronchial asthma.
After preparation made by attached sweet medicine, its appearance, color and other medicines preparation are approximate, and those skilled in the art are not easily distinguishable with appearance identification true and false, of poor quality.Prior art high performance liquid chromatography differentiates that the method for Herba Ephedrae, Radix Aconiti Lateralis Preparata and Radix Glycyrrhizae is numerous, it is experimentally confirmed the specificity for attached sweet pharmaceutical preparation all poor, therefore, in order to ensure attached sweet drug quality and clinical application effect, it is provided that a kind of discrimination method easy, reliable is extremely important.
Summary of the invention
The invention aims to provide the discrimination method of a kind of attached sweet medicine, for producing, circulate, use and checking, supervisory and management department a kind of discrimination method easy, reliable is provided.
The purpose of the present invention is achieved through the following technical solutions:
A kind of discrimination method of attached sweet medicine, it is characterised in that: adopt high effective liquid chromatography for measuring, use and connect, with polarity ether, the chromatographic column (being called for short: phenyl post) that phenyl bonded silica is filler.
The discrimination method of a kind of attached sweet medicine of the present invention, it is characterized in that: system suitability, with phosphoric acid solution=20 of acetonitrile 0.1% ~ 0.2% ~ 25 75 ~ 80 for mobile phase, detection wavelength is 222 ~ 242nm, and number of theoretical plate calculates by Benzoylmesaconine peak should be not less than 5000；
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.1% ~ 0.2%, shakes up, as solid-phase extraction column system suitability reference substance solution；Precision measures the above-mentioned each 3 ~ 30ml of reference substance stock solution, 1 ~ 5ml, 1 ~ 5ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.1% ~ 0.2%, shake up, prepare into every 1ml containing Benzoylmesaconine 3 ~ 30 μ g, benzoyl aconine 1 ~ 5 μ g, benzoyl hypo-aconine 1 ~ 5 μ g reference substance solution；
Solid-phase extraction column system suitability precision measures Benzoylmesaconine, benzoyl aconine, each 10ml of reference substance stock solution of benzoyl hypo-aconine, 5ml and 5ml, mixing, reduced pressure at room temperature is recycled to dry, dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml, precision measures 10ml, it is placed on the solid-phase extraction column handled well, this solid-phase extraction column exchanges reverse phase absorption agent for filler with mixed type cation, specification is 100 ~ 200mg, 4 ~ 8ml, in advance successively with acetonitrile, each 6ml eluting of water；Successively with water 3ml, the ammonia solution of 1.25%, water, methanol, each 5ml eluting of acetonitrile, after eluting liquid stream is most, place 5 minutes, then with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10, collect eluent, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds mobile phase 5ml makes dissolving, filters, takes subsequent filtrate as solid-phase extraction column system suitability solution；
Precision draws said system employment and suitability test (E & ST) solution and each 10 ~ 20 μ l of reference substance solution respectively, injects chromatograph of liquid, measures, the ratio of computing system employment and suitability test (E & ST) solution corresponding Component peak area each to reference substance solution, cannot be less than 0.95；
The preparation of need testing solution: take inspection product finely ground, weigh 0.2 ~ 0.6g, puts in tool plug conical flask, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, latter 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution；
Differentiate: draw reference substance solution and each 10 ~ 25 μ l of need testing solution respectively, inject hplc determination, test sample chromatograph presents the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time, namely determine containing Radix Aconiti Lateralis Preparata in preparation, otherwise without Radix Aconiti Lateralis Preparata in preparation.
A kind of discrimination method of the attached sweet medicine of the present invention, it is characterised in that: number of theoretical plate calculates by Benzoylmesaconine peak should be not less than 10000.
A kind of discrimination method of the attached sweet medicine of the present invention, it is characterised in that: when preparing need testing solution, sampling amount is 0.5g.
A kind of discrimination method of the attached sweet medicine of the present invention, it is characterized in that: in the preparation process of need testing solution, solid phase extraction column stuffing used is the exchange reverse phase absorption agent of mixed type cation, and specification is 100 ~ 200mg, volume is 4 ~ 8ml, and processes with acetonitrile, each 6ml eluting of water successively in advance.
A kind of discrimination method of the attached sweet medicine of the present invention, it is characterized in that: in the preparation process of need testing solution, the elution process of solid-phase extraction column is followed successively by water 3ml, the ammonia solution of 1.25%, water, methanol, each 5ml of acetonitrile, after eluting liquid stream is most, place 5 minutes, then with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10, collect eluent.
A kind of discrimination method of the attached sweet medicine of the present invention, it is characterized in that: differentiate Radix Glycyrrhizae by thin layer chromatography: take inspection product finely ground, weigh 0.5 ~ 2.5g, add diethyl ether 40ml, it is heated to reflux 1 hour, filtering, discard ether liquid, medicinal residues add methanol 30ml, it is heated to reflux 1 hour, filtering, filtrate is evaporated, and the residue 40ml that adds water makes dissolving, with n-butanol extraction 3 times, each 20ml, merges n-butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, n-butyl alcohol liquid is evaporated, and residue adds methanol 1ml makes dissolving, as need testing solution；Another extracting liquorice control medicinal material 0.5 ~ 1.5g, is made in the same way of control medicinal material solution；Extracting liquorice glycosides reference substance again, adds methanol and makes every 1ml solution containing 1.5 ~ 3mg, as reference substance solution；Draw each 3 ~ 10 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, taking out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, put and inspect under 365nm ultra-violet lamp, in test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color, namely determines it is the preparation containing Radix Glycyrrhizae.
The invention has the beneficial effects as follows: provide the discrimination method of the attached sweet medicine that system suitability is good, easy accurately, specificity is strong, ruggedness is good.Provide in the production of medicine, circulation, use procedure for relevant supervisory and management department and a kind of differentiate true and false, good and bad foundation.
In order to be better understood from the present invention, the methodological study below by way of discrimination method of the present invention further illustrates beneficial effects of the present invention.Methodological study is further intended to illustrate the effect of the present invention, and the restriction of non-invention.
One, sample is prepared
1, attached sweet granule is prepared
Taking Radix Aconiti Lateralis Preparata 15kg, Radix Glycyrrhizae 6kg, boiling twice, first time adds 8 times amount water, decocts 2 hours, and second time adds 6 times amount water, decoct 1 hour, merge twice decocting liquid, filter, concentration, drying under reduced pressure, pulverize, add dextrin appropriate, mixing, wet granulation, dry, granulate, obtain the granule of attached sweet medicine.
2, the negative granules agent of Radix Aconiti Lateralis Preparata it is prepared without
Extracting liquorice 300g, makes the negative granules agent without Radix Aconiti Lateralis Preparata according to the method preparing attached sweet granule.
3, the negative granules agent of Radix Glycyrrhizae it is prepared without
Take Radix Aconiti Lateralis Preparata 750g, make the negative granules agent without Radix Glycyrrhizae according to the method preparing attached sweet granule.
Two, Radix Aconiti Lateralis Preparata discrimination method and Method validation
1, solid-phase extraction column system suitability
Chromatographic condition and system suitability: be connected the chromatographic column that phenyl bonded silica is filler with polarity ether；With acetonitrile: the phosphoric acid solution of 0.14%=23 77 are for mobile phase；Detection wavelength is 232nm.Number of theoretical plate is calculated as 13054 by Benzoylmesaconine peak.
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.14%, shakes up, as solid-phase extraction column system suitability reference substance solution.
nullSolid-phase extraction column system suitability: precision measures Benzoylmesaconine、Benzoyl aconine、The each 10ml of reference substance stock solution of benzoyl hypo-aconine、5ml and 5ml，Mixing，Reduced pressure at room temperature is recycled to dry，Dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml，Precision measures 10ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，150mg，6ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with water 3ml，The ammonia solution of 1.25%、Water、Methanol、The each 5ml eluting of acetonitrile，After eluting liquid stream is most，Place 5 minutes，Again with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue adds mobile phase 5ml makes dissolving，Filter，Take subsequent filtrate as solid-phase extraction column system suitability solution.
Precision draws above-mentioned solid phase extraction column system suitability solution and each 10 ~ 20 μ l of reference substance solution respectively, injects chromatograph of liquid, measures.
Result: in solid-phase extraction column system suitability solution, Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine peak area are respectively: 541539,264615,304233, in reference substance solution, the peak area of corresponding composition is 546918,264040,307307 respectively；The ratio of corresponding Component peak area each to reference substance solution in solid-phase extraction column system suitability solution, is 0.99,1.00,0.99 respectively, and its ratio is all higher than 0.95, illustrates that this method solid-phase extraction column system suitability is good.
2, specificity
Prepared by reference substance solution: take Benzoylmesaconine under solid-phase extraction column system suitability item, benzoyl aconine, benzoyl hypo-aconine reference substance solution stock solution, precision measures 15ml, 2ml, 3ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.14%, shake up, prepare into every 1ml containing Benzoylmesaconine 15 μ g, benzoyl aconine 2 μ g, benzoyl hypo-aconine 3 μ g reference substance solution.
Prepared by need testing solution: take attached sweet granule finely ground, weigh 0.5g, puts in tool plug conical flask, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, latter 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution.
The preparation of negative need testing solution: take the negative preparation 0.5g without Radix Aconiti Lateralis Preparata, according to the negative need testing solution of need testing solution preparation method preparation.
Measure: take need testing solution, negative sample solution and reference substance solution, measure according to liquid-phase condition under solid-phase extraction column system suitability item and algoscopy.
Result, need testing solution chromatograph with reference substance solution chromatograph relevant position on have the chromatographic peak of identical retention time, and separating degree is good, and negative sample solution is to detecting without chromatographic peak with reference substance solution appearance time, explanation this method carries out Radix Aconiti Lateralis Preparata discriminating, and specificity is good.
3, ruggedness
3.1, stability of solution
Taking need testing solution room temperature to place, drew 20 μ l respectively at the 0th, 3,6,9,12 hours, inject chromatograph of liquid, measure, record chromatographic peak area, result shows that need testing solution is stable in 12 hours, all can Radix Aconiti Lateralis Preparata effectively be differentiated.
3.2, detection wavelength
Taking this product need testing solution, be measured respectively under 222nm, 232nm, 242nm wavelength, result shows that Radix Aconiti Lateralis Preparata all can effectively be differentiated by this method in this wave-length coverage, good tolerance.
3.3, flow velocity
Taking this product need testing solution, be respectively adopted 0.8ml/min, 1.0ml/min, 1.2ml/min flow velocity and be measured, result shows that Radix Aconiti Lateralis Preparata all can effectively be differentiated by this method in this flow rates, good tolerance.
3.4, mobile phase ratio adjustment
Taking this product need testing solution, the ratio being respectively adopted the phosphoric acid solution of acetonitrile 0.14% is that 20:80,23:77,25:75 are measured, it is shown that Radix Aconiti Lateralis Preparata all can effectively be differentiated by this method under above-mentioned three kinds of mobile phase ratios, and good tolerance.
3.5, column temperature
Take this product need testing solution, be respectively adopted 25 DEG C, 30 DEG C, 35 DEG C column temperatures and be measured, it is shown that Radix Aconiti Lateralis Preparata all can effectively be differentiated by this method within the scope of this column temperature, good tolerance.
3.6, the chromatographic column of different fillers
Take this product, be respectively adopted and connect phenyl bonded silica (hereinafter referred to as " phenyl post ") and octadecylsilane chemically bonded silica (hereinafter referred to as " C with polarity ether18Post ") measure for the chromatographic column of filler, the chromatographic column theoretical cam curve of two kinds of fillers and tailing factor are compared as follows:
Chromatographic column number of theoretical plate and the tailing factor of the different filler of table 1 compare
Being found out by result, two kinds of chromatographic column theoretical cam curve differences are little, but polarity ether connects phenyl bonded silica post glue and effectively reduces tailing factor, improves peak shape.Illustrate that polarity ether connects phenyl bonded silica post glue and is more suitable for this product mensuration.
Three, Radix Glycyrrhizae discrimination method and Method validation process
1, discrimination method
Employing thin layer chromatography differentiates.
Taking inspection product finely ground, weigh 2g, add diethyl ether 40ml, is heated to reflux 1 hour, filtering, discard ether liquid, medicinal residues add methanol 30ml, being heated to reflux 1 hour, filter, filtrate is evaporated, the residue 40ml that adds water makes dissolving, with n-butanol extraction 3 times, each 20ml, merge n-butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, n-butyl alcohol liquid is evaporated, and residue adds methanol 1ml makes dissolving, as need testing solution.Another extracting liquorice control medicinal material 1g, is made in the same way of control medicinal material solution.Extracting liquorice glycosides reference substance again, adds methanol and makes every 1ml solution containing 2mg, as reference substance solution.Draw each 5 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, taking out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, put and inspect under 365nm ultra-violet lamp.In test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color, namely determine it is the preparation containing Radix Glycyrrhizae.
2, Method validation
2.1, specificity
Take attached sweet granule 2g, prepare need testing solution according to said method.
Extracting liquorice control medicinal material 1g, is made in the same way of control medicinal material solution.
Extracting liquorice glycosides reference substance, adds methanol and makes every 1ml solution containing 2mg, as reference substance solution.
Take the negative preparation 2g without Radix Glycyrrhizae, according to the negative need testing solution of need testing solution preparation method preparation.
Draw each 5 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, taking out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, put and inspect under 365nm ultra-violet lamp.
As a result, in test sample chromatograph, the fluorescence speckle of aobvious same color on position corresponding with Radix Glycyrrhizae control medicinal material and liquirtin reference substance chromatograph；Negative sample solution is at relevant position immaculate.Illustrate that this law differentiates that Radix Glycyrrhizae specificity is good.
2.2, ruggedness
2.2.1, stability of solution
Taking need testing solution under specificity item, control medicinal material solution, reference substance solution and developing solvent, ambient temperatare was put, respectively at point sample, expansion in the 0th, 4,8 hours.It is good that result shows that above-mentioned each solution places 8 hours internal stabilities in room temperature, can effectively Radix Glycyrrhizae be differentiated..
2.2.2, temperature is launched
Take need testing solution, control medicinal material solution and reference substance solution point on same thin laminate, launch at 10 DEG C, 20 DEG C, 30 DEG C.
Result shows that the method is launched well, can effectively Radix Glycyrrhizae be differentiated within the scope of said temperature.
The present invention is further described below by specific embodiment, " the phenyl post " that specific embodiment uses uses the time for non-same model or same model difference, it is embodied in number of theoretical plate difference, but the number of theoretical plate of each collection of illustrative plates, tailing factor and separating degree etc. all meet regulation.
Detailed description of the invention
Embodiment 1: attached sweet granule differentiates
1) granule is prepared: take Radix Aconiti Lateralis Preparata 6kg, Radix Glycyrrhizae 1kg, boiling twice, first time adds 8 times amount water, decocts 2 hours, and second time adds 6 times amount water, decoct 1 hour, merge twice decocting liquid, filter, concentration, drying under reduced pressure, pulverize, and adds dextrin appropriate, mixing, wet granulation, dry, granulate, obtains the granule of attached sweet medicine.
2) Radix Aconiti Lateralis Preparata is differentiated by high performance liquid chromatography:
Chromatographic condition and system suitability: be connected the chromatographic column (WelchUlimatePhenyl-Ether, new post) that phenyl bonded silica is filler with polarity ether；With acetonitrile: the phosphoric acid solution of 0.1%=20 80 are for mobile phase；Detection wavelength is 222nm.Number of theoretical plate is calculated as 14106 by Benzoylmesaconine peak.
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.1%, shakes up, as solid-phase extraction column system suitability reference substance solution；Precision measures above-mentioned reference substance stock solution each 5ml, 1ml, 1ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.1%, shake up, prepare into every 1ml containing Benzoylmesaconine 5 μ g, benzoyl aconine 1 μ g, benzoyl hypo-aconine 1 μ g reference substance solution.
nullSolid-phase extraction column system suitability precision measures Benzoylmesaconine、Benzoyl aconine、The each 10ml of reference substance stock solution of benzoyl hypo-aconine、5ml and 5ml，Mixing，Reduced pressure at room temperature is recycled to dry，Dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml，Precision measures 10ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，100mg，4ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with water 3ml，The ammonia solution of 1.25%、Water、Methanol、The each 5ml eluting of acetonitrile，After eluting liquid stream is most，Place 5 minutes，Again with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue adds mobile phase 5ml makes dissolving，Filter，Take subsequent filtrate as solid-phase extraction column system suitability solution；
Precision draws said system employment and suitability test (E & ST) solution and each 10 μ l of reference substance solution respectively, injects chromatograph of liquid, measures, the ratio of computing system employment and suitability test (E & ST) solution corresponding Component peak area each to reference substance solution, cannot be less than 0.95.,
The preparation of need testing solution: take attached sweet granule finely ground, weigh 0.2g, puts in tool plug conical flask, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, latter 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution.
Discrimination method: draw reference substance solution and each 20 μ l of need testing solution respectively, inject hplc determination.
Result:
I: solid-phase extraction column system suitability solution and reference substance peak area and corresponding ratio are in Table 2.
Table 2 solid-phase extraction column system suitability result
| Benzoylmesaconine | Benzoyl aconine | Benzoyl hypo-aconine | |
| Reference substance solution | 251061 | 132029 | 150097 |
| Need testing solution | 246577 | 130708 | 145594 |
| Ratio | 0.98 | 0.99 | 0.97 |
II: identification result
Test sample chromatograph presents the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time.Conclusion: containing Radix Aconiti Lateralis Preparata in inspection product granule.
3) Radix Glycyrrhizae is differentiated by thin layer chromatography:
Taking attached sweet granule finely ground, weigh 2.5g, add diethyl ether 40ml, is heated to reflux 1 hour, filtering, discard ether liquid, medicinal residues add methanol 30ml, being heated to reflux 1 hour, filter, filtrate is evaporated, the residue 40ml that adds water makes dissolving, with n-butanol extraction 3 times, each 20ml, merge n-butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, n-butyl alcohol liquid is evaporated, and residue adds methanol 1ml makes dissolving, as need testing solution.Another extracting liquorice control medicinal material 1.5g, is made in the same way of control medicinal material solution.Extracting liquorice glycosides reference substance again, adds methanol and makes every 1ml solution containing 3mg, as reference substance solution.Draw each 3 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, taking out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, put and inspect under 365nm ultra-violet lamp.
Result: in test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color.Conclusion, containing Radix Glycyrrhizae in inspection product granule.
Embodiment 2: attached sweet granule differentiates
1) granule is prepared: take Radix Aconiti Lateralis Preparata 18kg, Radix Glycyrrhizae 9kg, boiling twice, first time adds 10 times amount water, decocts 1.5 hours, and second time adds 8 times amount water, decoct 1 hour, merge twice decocting liquid, filter, concentration, drying under reduced pressure, pulverize, and adds dextrin appropriate, mixing, wet granulation, dry, granulate, obtains the granule of attached sweet medicine.
2) Radix Aconiti Lateralis Preparata is differentiated by high performance liquid chromatography:
Chromatographic condition and system suitability: be connected the chromatographic column (WelchUlimatePhenyl-Ether uses 2 months) that phenyl bonded silica is filler with polarity ether；With acetonitrile: the phosphoric acid solution of 0.14%=23 77 are for mobile phase；Detection wavelength is 232nm.Number of theoretical plate is calculated as 10141 by Benzoylmesaconine peak.
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.14%, shakes up, as solid-phase extraction column system suitability reference substance solution；Precision measures above-mentioned reference substance stock solution each 15ml, 2ml, 2ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.14%, shake up, prepare into every 1ml containing Benzoylmesaconine 15 μ g, benzoyl aconine 2 μ g, benzoyl hypo-aconine 2 μ g reference substance solution；
nullSolid-phase extraction column system suitability precision measures Benzoylmesaconine、Benzoyl aconine、The each 10ml of reference substance stock solution of benzoyl hypo-aconine、5ml and 5ml，Mixing，Reduced pressure at room temperature is recycled to dry，Dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml，Precision measures 10ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，200mg，8ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with water 3ml，The ammonia solution of 1.25%、Water、Methanol、The each 5ml eluting of acetonitrile，After eluting liquid stream is most，Place 5 minutes，Again with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue adds mobile phase 5ml makes dissolving，Filter，Take subsequent filtrate as solid-phase extraction column system suitability solution；
Precision draws said system employment and suitability test (E & ST) solution and each 15 μ l of reference substance solution respectively, injects chromatograph of liquid, measures, the ratio of computing system employment and suitability test (E & ST) solution corresponding Component peak area each to reference substance solution, is all higher than 0.95.
The preparation of need testing solution: take attached sweet granule finely ground, weigh 0.6g, puts in tool plug conical flask, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, latter 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution.
Discrimination method: draw reference substance solution and each 10 μ l of need testing solution respectively, inject chromatograph of liquid, measure.
Result: present the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time in test sample chromatograph, conclusion: containing Radix Aconiti Lateralis Preparata in inspection product granule.
3) Radix Glycyrrhizae is differentiated by thin layer chromatography:
Taking attached sweet granule finely ground, weigh 0.5g, add diethyl ether 40ml, is heated to reflux 1 hour, filtering, discard ether liquid, medicinal residues add methanol 30ml, being heated to reflux 1 hour, filter, filtrate is evaporated, the residue 40ml that adds water makes dissolving, with n-butanol extraction 3 times, each 20ml, merge n-butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, n-butyl alcohol liquid is evaporated, and residue adds methanol 1ml makes dissolving, as need testing solution；Another extracting liquorice control medicinal material 0.5g, is made in the same way of control medicinal material solution；Extracting liquorice glycosides reference substance again, adds methanol and makes every 1ml solution containing 1.5mg, as reference substance solution；Draw each 10 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, taking out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, put and inspect under 365nm ultra-violet lamp.
Result: in test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color.Conclusion: containing Radix Glycyrrhizae in inspection product granule.
Embodiment 3: attached sweet granule differentiates
1) granule is prepared: taking Radix Aconiti Lateralis Preparata 10kg, Radix Glycyrrhizae 5kg, boiling three times, first time adds 8 times amount water, decocting 1.5 hours, second time adds 8 times amount water, decocts 1 hour, third time adds 6 times amount water, decocts 1 hour, merges three decocting liquids, filter, concentration, drying under reduced pressure, pulverize, add dextrin appropriate, mixing, wet granulation, dry, granulate, obtain the granule of attached sweet medicine.
2) Radix Aconiti Lateralis Preparata is differentiated by high performance liquid chromatography:
Chromatographic condition and system suitability: be connected the chromatographic column (VenusilXBPPolar-Phenyl, new post) that phenyl bonded silica is filler with polarity ether；With acetonitrile: the phosphoric acid solution of 0.2%=25 75 are for mobile phase；Detection wavelength is 242nm.Number of theoretical plate is calculated as 14065 by Benzoylmesaconine peak.
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.2%, shakes up, as solid-phase extraction column system suitability reference substance solution；Precision measures above-mentioned reference substance stock solution each 30ml, 5ml, 5ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.2%, shake up, prepare into every 1ml containing Benzoylmesaconine 30 μ g, benzoyl aconine 5 μ g, benzoyl hypo-aconine 5 μ g reference substance solution.
nullSolid-phase extraction column system suitability precision measures Benzoylmesaconine、Benzoyl aconine、The each 10ml of reference substance stock solution of benzoyl hypo-aconine、5ml and 5ml，Mixing，Reduced pressure at room temperature is recycled to dry，Dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml，Precision measures 10ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，150mg，6ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with water 3ml，The ammonia solution of 1.25%、Water、Methanol、The each 5ml eluting of acetonitrile，After eluting liquid stream is most，Place 5 minutes，Again with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue adds mobile phase 5ml makes dissolving，Filter，Take subsequent filtrate as solid-phase extraction column system suitability solution；
Precision draws said system employment and suitability test (E & ST) solution and each 20 μ l of reference substance solution respectively, injects chromatograph of liquid, measures, the ratio of computing system employment and suitability test (E & ST) solution corresponding Component peak area each to reference substance solution, is all higher than 0.95.
The preparation of need testing solution: take attached sweet granule finely ground, weigh 0.4g, puts in tool plug conical flask, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, latter 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution；
Discrimination method: draw reference substance solution and each 15 μ l of need testing solution respectively, inject chromatograph of liquid, measure.
Result: present the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time in test sample chromatograph, conclusion: containing Radix Aconiti Lateralis Preparata in inspection product granule.
3) Radix Glycyrrhizae is differentiated by thin layer chromatography:
Taking attached sweet granule finely ground, weigh 1.5g, add diethyl ether 40ml, is heated to reflux 1 hour, filtering, discard ether liquid, medicinal residues add methanol 30ml, being heated to reflux 1 hour, filter, filtrate is evaporated, the residue 40ml that adds water makes dissolving, with n-butanol extraction 3 times, each 20ml, merge n-butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, n-butyl alcohol liquid is evaporated, and residue adds methanol 1ml makes dissolving, as need testing solution；Another extracting liquorice control medicinal material 1g, is made in the same way of control medicinal material solution；Extracting liquorice glycosides reference substance again, adds methanol and makes every 1ml solution containing 3mg, as reference substance solution；Draw each 5 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, taking out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, put and inspect under 365nm ultra-violet lamp.
Result: in test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color.Conclusion: containing Radix Glycyrrhizae in inspection product granule.
Embodiment 4: attached sweet tablet differentiates
1) tablet is prepared: take Radix Aconiti Lateralis Preparata 10kg, Radix Glycyrrhizae 4kg, boiling 3 times.First time adds the water of 10 times amount of crude drug quality, decocts 2h, and second and third time respectively adds the water of 8 times amount respectively, decoct 1.5h respectively, merge 3 decoction liquor, filter, take filtrate reduced in volume and be 1.12(60 DEG C to relative density), add ethanol, just add just stirring and evenly mixing, make alcohol content reach 70%, stand 24h, filter, filtrate recycling ethanol, be evaporated to relative density and be 1.25(60 DEG C), less than 60 DEG C drying under reduced pressure, pulverize, add starch in right amount, granulate, add Pulvis Talci appropriate, mixing, tabletting, obtain the tablet of attached sweet medicine.
2) Radix Aconiti Lateralis Preparata is differentiated by high performance liquid chromatography:
Chromatographic condition and system suitability: be connected the chromatographic column (VenusilXBPPolar-Phenyl uses 6 months) that phenyl bonded silica is filler with polarity ether；With acetonitrile: the phosphoric acid solution of 0.15%=23 77 are for mobile phase；Detection wavelength is 232nm；Number of theoretical plate is calculated as 5906 by Benzoylmesaconine peak.
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.15%, shakes up, as solid-phase extraction column system suitability reference substance solution；Precision measures above-mentioned reference substance stock solution each 15ml, 2ml, 4ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.15%, shake up, prepare into every 1ml containing Benzoylmesaconine 15 μ g, benzoyl aconine 2 μ g, benzoyl hypo-aconine 4 μ g reference substance solution.
nullSolid-phase extraction column system suitability precision measures Benzoylmesaconine、Benzoyl aconine、The each 10ml of reference substance stock solution of benzoyl hypo-aconine、5ml and 5ml，Mixing，Reduced pressure at room temperature is recycled to dry，Dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml，Precision measures 10ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，150mg，6ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with water 3ml，The ammonia solution of 1.25%、Water、Methanol、The each 5ml eluting of acetonitrile，After eluting liquid stream is most，Place 5 minutes，Again with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue adds mobile phase 5ml makes dissolving，Filter，Take subsequent filtrate as solid-phase extraction column system suitability solution；
Precision draws said system employment and suitability test (E & ST) solution and each 10 μ l of reference substance solution respectively, injects chromatograph of liquid, measures, the ratio of computing system employment and suitability test (E & ST) solution corresponding Component peak area each to reference substance solution, is all higher than 0.95.
The preparation of need testing solution: take attached sweet finely ground, weigh 0.4g, put tool plug conical flask in, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, latter 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution.
Discrimination method: draw reference substance solution and each 20 μ l of need testing solution respectively, inject chromatograph of liquid, measure.
Result: present the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time in test sample chromatograph, conclusion: containing Radix Aconiti Lateralis Preparata in attached sweet of product of inspection.
3) Radix Glycyrrhizae is differentiated by thin layer chromatography:
Take attached sweet finely ground, weigh 1.4g, add diethyl ether 40ml, is heated to reflux 1 hour, filtering, discard ether liquid, medicinal residues add methanol 30ml, being heated to reflux 1 hour, filter, filtrate is evaporated, the residue 40ml that adds water makes dissolving, with n-butanol extraction 3 times, each 20ml, merge n-butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, n-butyl alcohol liquid is evaporated, and residue adds methanol 1ml makes dissolving, as need testing solution；Another extracting liquorice control medicinal material 1g, is made in the same way of control medicinal material solution；Extracting liquorice glycosides reference substance again, adds methanol and makes every 1ml solution containing 2.5mg, as reference substance solution；Draw each 5 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, taking out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, put and inspect under 365nm ultra-violet lamp.
Result: in test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color.Conclusion: containing Radix Glycyrrhizae in attached sweet of product of inspection.
Embodiment 5: attached sweet capsule differentiates
1) capsule is prepared: take Radix Aconiti Lateralis Preparata 18kg, Radix Glycyrrhizae 6kg, boiling twice, first time adds 8 times amount water, decoct 2 hours, second time adds 6 times amount water, decoct 1 hour, merge twice decocting liquid, filter, take filtrate reduced in volume and be 1.15(60 DEG C to relative density), add ethanol, just just stirring and evenly mixing is added, alcohol content is made to reach 80%, stand 12h, filter, filtrate recycling ethanol, it is evaporated to relative density and is 1.2(60 DEG C), less than 60 DEG C drying under reduced pressure, pulverize, add micropowder silica gel appropriate, granulate, drying under reduced pressure, load capsule, obtain the capsule of attached sweet medicine.
2) Radix Aconiti Lateralis Preparata is differentiated by high performance liquid chromatography:
Chromatographic condition is connected, with polarity ether, the chromatographic column (WelchUlimateXB-Phenyl uses 1 month) that phenyl bonded silica is filler with system suitability；With acetonitrile: the phosphoric acid solution of 0.12%=22 78 are for mobile phase；Detection wavelength is 230nm；Number of theoretical plate is calculated as 12004 by Benzoylmesaconine peak.
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.12%, shakes up, as solid-phase extraction column system suitability reference substance solution；Precision measures above-mentioned reference substance stock solution each 20ml, 3ml, 5ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.12%, shake up, prepare into every 1ml containing Benzoylmesaconine 20 μ g, benzoyl aconine 3 μ g, benzoyl hypo-aconine 5 μ g reference substance solution.
nullSolid-phase extraction column system suitability precision measures Benzoylmesaconine、Benzoyl aconine、The each 10ml of reference substance stock solution of benzoyl hypo-aconine、5ml and 5ml，Mixing，Reduced pressure at room temperature is recycled to dry，Dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml，Precision measures 10ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，100mg，4ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with water 3ml，The ammonia solution of 1.25%、Water、Methanol、The each 5ml eluting of acetonitrile，After eluting liquid stream is most，Place 5 minutes，Again with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue adds mobile phase 5ml makes dissolving，Filter，Take subsequent filtrate as solid-phase extraction column system suitability solution；
Precision draws said system employment and suitability test (E & ST) solution and each 15 μ l of reference substance solution respectively, injects chromatograph of liquid, measures, the ratio of computing system employment and suitability test (E & ST) solution corresponding Component peak area each to reference substance solution, is all higher than 0.95.
The preparation of need testing solution: take this product content, weigh 0.3g, puts in tool plug conical flask, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, latter 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution.
Discrimination method: draw reference substance solution and each 15 μ l of need testing solution respectively, inject chromatograph of liquid, measure.
Result: present the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time in test sample chromatograph, conclusion: containing Radix Aconiti Lateralis Preparata in the inspection attached sweet capsule of product.
3) Radix Glycyrrhizae is differentiated by thin layer chromatography:
Taking this product content 1g, add diethyl ether 40ml, is heated to reflux 1 hour, filtering, discard ether liquid, medicinal residues add methanol 30ml, being heated to reflux 1 hour, filter, filtrate is evaporated, the residue 40ml that adds water makes dissolving, with n-butanol extraction 3 times, each 20ml, merge n-butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, n-butyl alcohol liquid is evaporated, and residue adds methanol 1ml makes dissolving, as need testing solution.Another extracting liquorice control medicinal material 1g, is made in the same way of control medicinal material solution.Extracting liquorice glycosides reference substance again, adds methanol and makes every 1ml solution containing 2mg, as reference substance solution.According to one the annex VIB thin layer chromatography test of " Chinese Pharmacopoeia " version in 2010, draw each 5 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, puts and inspects under 365nm ultra-violet lamp.
Result: in test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color.Conclusion: containing Radix Glycyrrhizae in the inspection attached sweet capsule of product.
Embodiment 6: attached sweet oral liquid differentiates
1) oral solutions is prepared: taking Radix Aconiti Lateralis Preparata 3kg, Radix Glycyrrhizae 1.2kg, boiling twice, first time adds 8 times amount water, decocting 2 hours, second time adds 6 times amount water, decocts 1 hour, merges twice decocting liquid, filter, take filtrate reduced in volume and be 1.12(60 DEG C to relative density), add ethanol, just add just stirring and evenly mixing, make alcohol content reach 75%, stand 24h, filter, filtrate recycling ethanol, concentration, it is appropriate to add sucrose and antibacterial, boils, constant volume, filter, fill, obtain the oral solutions of attached sweet medicine.
2) high performance liquid chromatography differentiates Radix Aconiti Lateralis Preparata:
Chromatographic condition is connected, with polarity ether, the chromatographic column (WelchUlimateXB-Phenyl uses 4 months) that phenyl bonded silica is filler with system suitability；With acetonitrile: the phosphoric acid solution of 0.16%=24 76 are for mobile phase；Detection wavelength is 237nm.Number of theoretical plate is calculated as 7480 by Benzoylmesaconine peak.
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.16%, shakes up, as solid-phase extraction column system suitability reference substance solution；Precision measures above-mentioned reference substance stock solution each 17ml, 4ml, 3ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.16%, shake up, prepare into every 1ml containing Benzoylmesaconine 17 μ g, benzoyl aconine 4 μ g, benzoyl hypo-aconine 3 μ g reference substance solution.
nullSolid-phase extraction column system suitability precision measures Benzoylmesaconine、Benzoyl aconine、The each 10ml of reference substance stock solution of benzoyl hypo-aconine、5ml and 5ml，Mixing，Reduced pressure at room temperature is recycled to dry，Dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml，Precision measures 10ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，200mg，8ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with water 3ml，The ammonia solution of 1.25%、Water、Methanol、The each 5ml eluting of acetonitrile，After eluting liquid stream is most，Place 5 minutes，Again with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue adds mobile phase 5ml makes dissolving，Filter，Take subsequent filtrate as solid-phase extraction column system suitability solution；
Precision draws said system employment and suitability test (E & ST) solution and each 10 μ l of reference substance solution respectively, injects chromatograph of liquid, measures, the ratio of computing system employment and suitability test (E & ST) solution corresponding Component peak area each to reference substance solution, is all higher than 0.95.
The preparation of need testing solution: measure attached sweet oral liquid 3ml, put in tool plug conical flask, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution.
Discrimination method: draw reference substance solution and each 10 μ l of need testing solution respectively, inject chromatograph of liquid, measure.
Result: present the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time in test sample chromatograph, illustrates that inspection is savored in oral liquid containing Radix Aconiti Lateralis Preparata.
3) Radix Glycyrrhizae is differentiated by thin layer chromatography:
Measure this product content 10ml, add methanol 25ml, shake up, supersound process 10min, filters, and filtrate is evaporated, residue add water 10ml dissolve, add diethyl ether 20ml, extracts 1 time, discard ether liquid, water liquid n-butanol extraction 3 times, each 20ml, merge n-butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, n-butyl alcohol liquid is evaporated, and residue adds methanol 1ml makes dissolving, as need testing solution.Another extracting liquorice control medicinal material 1g, add diethyl ether 40ml, is heated to reflux 1 hour, filtering, discard ether liquid, medicinal residues add methanol 30ml, it is heated to reflux 1 hour, filtering, filtrate is evaporated, and the residue 40ml that adds water makes dissolving, according to need testing solution preparation method, operate after " using n-butanol extraction 3 times, each 20ml " in accordance with the law, make control medicinal material solution.Extracting liquorice glycosides reference substance again, adds methanol and makes every 1ml solution containing 2mg, as reference substance solution.According to one the annex VIB thin layer chromatography test of " Chinese Pharmacopoeia " version in 2010, draw each 5 μ l of above-mentioned four kinds of solution, put respectively on silica gel g thin-layer plate prepared by same use 1% sodium hydroxide solution, with n-butyl alcohol strong ammonia solution ethanol=5 21 for developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, clear to spot development 105 DEG C of heating, puts and inspects under 365nm ultra-violet lamp.
Result: in test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color.Conclusion: containing Radix Glycyrrhizae in the inspection attached sweet oral liquid of product.
Claims (3)
1. the discrimination method of an attached sweet medicine, it is characterised in that: differentiating Radix Aconiti Lateralis Preparata by high performance liquid chromatography, its chromatographic column connects phenyl bonded silica for filler with polarity ether；System suitability, with phosphoric acid solution=20 of acetonitrile 0.1% ~ 0.2% ~ 25 75 ~ 80 for mobile phase, detection wavelength is 222 ~ 242nm, and number of theoretical plate calculates by Benzoylmesaconine peak should be not less than 5000；
The preparation of reference substance solution takes Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures the above-mentioned each 10ml of reference substance stock solution, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale with the phosphoric acid solution of 0.1% ~ 0.2%, shakes up, as solid-phase extraction column system suitability reference substance solution；Precision measures the above-mentioned each 3 ~ 30ml of reference substance stock solution, 1 ~ 5ml, 1 ~ 5ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, it is diluted to scale with the phosphoric acid solution of 0.1% ~ 0.2%, shake up, prepare into every 1ml containing Benzoylmesaconine 3 ~ 30 μ g, benzoyl aconine 1 ~ 5 μ g, benzoyl hypo-aconine 1 ~ 5 μ g reference substance solution；
Solid-phase extraction column system suitability precision measures Benzoylmesaconine, benzoyl aconine, each 10ml of reference substance stock solution of benzoyl hypo-aconine, 5ml and 5ml, mixing, reduced pressure at room temperature is recycled to dry, dissolve in right amount with 0.1mol/L hydrochloric acid solution and be settled to 200ml, precision measures 10ml, it is placed on the solid-phase extraction column handled well, this solid-phase extraction column exchanges reverse phase absorption agent for filler with mixed type cation, specification is 100 ~ 200mg, 4 ~ 8ml, in advance successively with acetonitrile, each 6ml eluting of water；Successively with water 3ml, the ammonia solution of 1.25%, water, methanol, each 5ml eluting of acetonitrile, after eluting liquid stream is most, place 5 minutes, then with the mixed solution 10ml eluting of acetonitrile strong ammonia solution=90 10, collect eluent, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds mobile phase 5ml makes dissolving, filters, takes subsequent filtrate as solid-phase extraction column system suitability solution；
Precision draws said system employment and suitability test (E & ST) solution and each 10 ~ 20 μ l of reference substance solution respectively, injects chromatograph of liquid, measures, the ratio of computing system employment and suitability test (E & ST) solution corresponding Component peak area each to reference substance solution, cannot be less than 0.95；
The preparation of need testing solution: take inspection product finely ground, weigh 0.2 ~ 0.6g, puts in tool plug conical flask, add 0.1mol/L hydrochloric acid solution 25ml, close plug, supersound process 40 minutes jolting constantly, latter 4000 revs/min are centrifuged 30 minutes, filter, measure subsequent filtrate 10ml, according to solid-phase extraction column system suitability, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, prepare need testing solution；
Differentiate: draw reference substance solution and each 10 ~ 25 μ l of need testing solution respectively, inject hplc determination, test sample chromatograph presents the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time, namely determine containing Radix Aconiti Lateralis Preparata in preparation, otherwise without Radix Aconiti Lateralis Preparata in preparation.
2. the discrimination method of a kind of attached sweet medicine according to claim 1, it is characterised in that: number of theoretical plate calculates by Benzoylmesaconine peak should be not less than 10000.
3. the discrimination method of a kind of attached sweet medicine according to claim 1, it is characterised in that: when preparing need testing solution, sampling amount is 0.5g.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510241416.7A CN104820055B (en) | 2015-05-13 | 2015-05-13 | A kind of discrimination method of attached sweet medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510241416.7A CN104820055B (en) | 2015-05-13 | 2015-05-13 | A kind of discrimination method of attached sweet medicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104820055A CN104820055A (en) | 2015-08-05 |
| CN104820055B true CN104820055B (en) | 2016-07-13 |
Family
ID=53730410
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510241416.7A Active CN104820055B (en) | 2015-05-13 | 2015-05-13 | A kind of discrimination method of attached sweet medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104820055B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110794079A (en) * | 2019-11-20 | 2020-02-14 | 刘圣梅 | Identification method of ephedra and aconitine preparation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101537088A (en) * | 2009-04-14 | 2009-09-23 | 文永盛 | Method for controlling quality of Longdanxiegan Capsule |
| CN102520109A (en) * | 2011-12-06 | 2012-06-27 | 西安泰科迈医药科技有限公司 | Method for detecting quality of cane sugar-free sleep-benefiting brain-nourishing syrup |
-
2015
- 2015-05-13 CN CN201510241416.7A patent/CN104820055B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101537088A (en) * | 2009-04-14 | 2009-09-23 | 文永盛 | Method for controlling quality of Longdanxiegan Capsule |
| CN102520109A (en) * | 2011-12-06 | 2012-06-27 | 西安泰科迈医药科技有限公司 | Method for detecting quality of cane sugar-free sleep-benefiting brain-nourishing syrup |
Non-Patent Citations (1)
| Title |
|---|
| Separation Methods for Toxic Components in Traditional Chinese Medicines;Wei Li等;《Analytical Sciences》;20050930;第21卷;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104820055A (en) | 2015-08-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112666268B (en) | Method for identifying various components and measuring content of traditional Chinese medicine composition | |
| CN105259295B (en) | Quality detection method for ginseng, cassia twig and poria cocos oral solution | |
| CN102269751B (en) | Detection method of Liuweinengxiao preparation | |
| CN104502518B (en) | A kind of detection method for the treatment of the Chinese medicine preparation of baby anorexia | |
| CN104161847B (en) | A kind of quality determining method of the Chinese medicine composition treating diabetic retinopathy | |
| CN101537088B (en) | Method for controlling quality of Longdanxiegan Capsule | |
| CN104849369B (en) | The attached sweet drug quality detection method of a kind of fiber crops | |
| CN104792911B (en) | A kind of discrimination method of the attached sweet preparation of fiber crops | |
| CN105301168B (en) | The detection method of dredging collateral resolving sputum capsule | |
| CN102335402A (en) | Detection method of Chinese preparation mixture for invigorating the spleen and replenishing qi | |
| CN103344737A (en) | Quality control method of traditional Chinese medicine tablet for treating nasosinusitis | |
| CN104407092A (en) | Quality detection method of traditional Chinese medicinal composition with efficacy of appetizing and invigorating spleen | |
| CN102139039A (en) | Quality control method of coptis tablet for clearing away stomach heat | |
| CN104833754B (en) | A kind of attached sweet drug detection method | |
| CN103585204B (en) | Thesium granule with high stability and preparation method thereof | |
| CN104820055B (en) | A kind of discrimination method of attached sweet medicine | |
| CN102139040A (en) | Quality control method of tablet capable of clearing lung and inhibiting fire | |
| CN101961472B (en) | Detection method for lung-soothing syrup | |
| CN104111295A (en) | Method for controlling quality of Chinese herbal preparation | |
| CN103267824A (en) | Method for detecting wind-cold cold granules | |
| CN114113425A (en) | Method for identifying cortex phellodendri chinensis in radix scutellariae and rhizoma coptidis preparation to replace cortex phellodendri chinensis in medicine by using high performance liquid chromatography | |
| CN100484558C (en) | Ginseng freeze-drying powdery injection and its quality control method | |
| CN102552751A (en) | Quality detection method for medicine (named as Er bao concentrated decoction) | |
| CN104833756A (en) | Method for determining content of mono-ester type alkaloids in monkshood-radix glycyrrhizae medicament | |
| CN101417081B (en) | Quality detection method of Longfengbao capsule |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: 250014 81 thousand Buddha Shandong Road, Lixia District, Ji'nan, Shandong Patentee after: Shandong Zhonghong Kang Pharmaceutical Technology Development Co Ltd Address before: 250014 No. 4, West building, Shandong Academy of Sciences, 19 Shandong Road, Lixia Road, Lixia District, Shandong Patentee before: Jinan Kangzhong Medicin Science & Technology Co., Ltd. |
|
| CP03 | Change of name, title or address |