CN104833754B - A kind of attached sweet drug detection method - Google Patents
A kind of attached sweet drug detection method Download PDFInfo
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Abstract
The invention provides a kind of attached sweet drug quality detection method, it is characterized in that: adopting and connect, with polarity ether, the chromatographic column that phenyl bonded silica is filler and differentiate and measure Radix Aconiti Lateralis Preparata content, its target component separating degree, peak shape, tailing factor have all had and significantly improve；Thin layer chromatography is adopted to differentiate Radix Glycyrrhizae；High performance liquid chromatography is adopted to control diester-type alkaloids content；Through related methodology checking and case expedition, it was shown that energy simplicity of the present invention differentiates attached sweet medicine, control diester-type alkaloids content, detection Radix Aconiti Lateralis Preparata and Radix Glycyrrhizae content accurately, in producing and using, effectively ensure product quality, and then ensure pharmaceutical effectiveness.
Description
Technical field
The present invention relates to a kind of attached sweet drug quality detection method, 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 9-1 part is that raw material makes (called after " attached sweet medicine "), has good curative effect for allergic rhinitis and bronchial asthma.
Allergic rhinitis and bronchial asthma are the closely-related respiratory system disease of anaphylaxis.Belong to frequently-occurring disease, difficult disease, and prevalence has the trend increased year by year.The drug main of existing treatment allergic rhinitis to have antihistaminic, glucocorticoids, anti-leukotriene medicine, anticholinergic and adrenomimetic etc., mainly nasal-cavity administration, life-time service is easily generated side effect and drug resistance, and recurrence rate after healing is high, and long-term efficacy is poor.In addition desensitization immunization therapy can not large area clinical expansion owing to the reason such as the length course for the treatment of is restricted.The research of this type of medicine is mainly the modified form of existing medicine both at home and abroad at present, it is important that the long-acting dosage form of spray, inhalant, to reduce times for spraying, reduces side effect, improves the compliance of patient.The feature of above-mentioned chemical medicine is rapid-action, and effect is obvious, but long-term effect is poor, it is impossible to radical cure, is easily generated drug resistance and side effect.
The existing clinical practice of the present invention it is shown that this product to allergic rhinitis and the determined curative effect of bronchial asthma, long-term effect is good, have no untoward reaction, compensate for a certain extent chemical medicine only control symptom, long-term effect difference etc. defect.
The prescription of attached sweet medicine contains Radix Aconiti Lateralis Preparata, and alkaloid contained by Radix Aconiti Lateralis Preparata mainly has di esters alkaloid, monoesters Alkaloid and amido alkaloid.Wherein di esters alkaloid toxicity is maximum, belongs to the liposoluble constituent being insoluble in water；Monoesters Alkaloid belongs to hydrophilic composition, and toxicity is little, is only di esters alkaloidal 1/200；Amido alkaloid belongs to strongly hydrophilic composition, and toxicity is very micro-, is only di esters alkaloidal 1/2000～1/4000.In Radix Aconiti Lateralis Preparata, severe toxicity composition is that diester-type alkaloids is mainly aconitine, mesaconitine and hypaconitine.Oral di esters alkaloid 3～4mg can the causing death (LD of studies on acute toxicity50For 11.8mg/Kg), illustrate that di esters alkaloid toxicity is extremely strong, it is the main cause that clinical practice is limited.
Attached sweet medicine of the present invention, prior art does not have the quality determining method of preparation, it is impossible to be effectively ensured product quality in producing and using, therefore, in order to ensure attached sweet drug quality and clinical application effect and safety, it is provided that a set of simplicity, reliable quality determining method are extremely important.
Summary of the invention
It is an object of the invention to provide the quality determining method of a kind of attached sweet medicine, it is determined that the limit of active constituent content and toxic component (dibasic acid esters alkaloid), to ensure Drug safety, effectiveness and quality controllability.
The purpose of the present invention is achieved through the following technical solutions:
The quality determining method of a kind of attached sweet medicine of the present invention, it is characterised in that:
[character] this product content be brown color to sepia, the micro-hardship sweet, micro-of taste；
[discriminating] (1) Radix Aconiti Lateralis Preparata differentiates: according to the method test under Radix Aconiti Lateralis Preparata assay item, should present the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time in test sample chromatograph；
(2) Radix Glycyrrhizae differentiates: taking this product content 0.5～2.5g, 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, wash with water 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；Test according to thin layer chromatography (one annex VI B of " Chinese Pharmacopoeia " version in 2010), draw each 5 μ l of above-mentioned three 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 2 1) 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 ultra-violet lamp (365nm)；In test sample chromatograph, on position corresponding with control medicinal material and reference substance chromatograph, the fluorescence speckle of aobvious same color；
[inspection] diester-type alkaloids: measure according to high performance liquid chromatography (one annex VI D of " Chinese Pharmacopoeia " version in 2010)；
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica for filler；With acetonitrile-oxolane (25 15) for mobile phase A, with 0.1% phosphoric acid solution containing 0.03mol/L potassium dihydrogen phosphate for Mobile phase B, the regulation according to the form below carries out gradient elution, and detection wavelength is 232nm；Number of theoretical plate calculates by aconitine peak should be not less than 30000；
Table 1 gradient elution program
It is accurately weighed in right amount that the preparation of reference substance solution takes mesaconitine reference substance, hypaconitine reference substance, aconitine reference substance, adds acetonitrile respectively and makes every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures each 5ml of reference substance stock solution, puts in 50ml measuring bottle, adds 0.1% phosphoric acid solution and is diluted to scale, shakes up, to obtain final product；
nullSolid-phase extraction column system suitability precision measures each 3ml of reference substance stock solution，Mixing，Reduced pressure at room temperature is recycled to dry，Residue precision adds 0.1mol/L hydrochloric acid solution 50ml makes dissolving，Precision measures 5ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，150mg，Capacity is 6ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with 0.lmol/L hydrochloric acid solution、Methanol、The each 5ml eluting of acetonitrile，Discard eluent，Place 5 minutes，Continue the mixed solution 10ml eluting with acetonitrile-strong ammonia solution (90 10)，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue precision adds the mixed solution 3ml of acetonitrile-0.1% phosphoric acid solution (30 70) 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 μ l of reference substance solution respectively, injects chromatograph of liquid, mensuration, computing system employment and suitability test (E & ST) solution and each corresponding Component peak area ratio of reference substance solution, cannot be less than 0.95；
The preparation of need testing solution: take this product appropriate, finely ground, take 1～3g, accurately weighed, put in tool plug conical flask, the accurate mixed solution 50ml adding acetonitrile-strong ammonia solution (90 10), close plug, weighed weight, supersound process 30 minutes, the weight of less loss is supplied with the mixed solution of acetonitrile-strong ammonia solution (90 10), shake up, filter, precision measures 25ml subsequent filtrate in less than 40 DEG C decompression and solvent recoveries to dry, residue precision adds 0.1mol/L hydrochloric acid solution 10ml makes dissolving, filter, filtrate is according to the method under solid-phase extraction column system suitability item, from " being placed on the solid-phase extraction column handled well ", operate in accordance with the law, obtain；
Algoscopy: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, to obtain final product；
Other should meet every regulation relevant under one annex rules of preparations item of " Chinese Pharmacopoeia " version in 2010；
[assay] Radix Aconiti Lateralis Preparata assay: measure according to high performance liquid chromatography (one annex VI D of " Chinese Pharmacopoeia " version in 2010)；
Chromatographic condition and system suitability: be connected the chromatographic column that phenyl bonded silica is filler with polarity ether；With the phosphoric acid solution (23 77) of acetonitrile-0.1% for mobile phase；Detection wavelength is 232nm, and number of theoretical plate calculates by Benzoylmesaconine peak and is 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, put in the measuring bottle of same 100ml, it is diluted to scale with the phosphoric acid solution of 0.1%, shake up, as solid-phase extraction column system suitability reference substance solution, precision measures each reference substance stock solution 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 0.1%, 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；
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 not less than 0.95；
The preparation of need testing solution takes attached sweet granule, finely ground, weigh 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 0.1mol/L hydrochloric acid solution 25ml, close plug, weighed weight, supersound process 40 minutes jolting constantly, let cool, more weighed weight, the weight of less loss is supplied with 0.1mol/L hydrochloric acid solution, shake up, leaving the heart 30 minutes with per minute 4000, filter, precision measures subsequent filtrate 10ml, according to the method under solid-phase extraction column system suitability item, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, to obtain final product；
Algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, measures, to obtain final product；
Radix Glycyrrhizae assay: measure according to high performance liquid chromatography (one annex VI D of " Chinese Pharmacopoeia " version in 2010)；
Chromatographic condition and system suitability: the chromatographic column being filler with octadecylsilane chemically bonded silica；With acetonitrile-0.5% glacial acetic acid solution (18 82) for mobile phase；Detection wavelength is 276nm；Number of theoretical plate calculates by liquirtin peak should be not less than 4000；
The preparation of reference substance solution: extracting liquorice glycosides reference substance is appropriate, accurately weighed, adds 70% ethanol and makes every 1ml solution containing 20 μ g, to obtain final product；
The preparation of need testing solution: take this product content, finely ground, take 0.2～0.6g, accurately weighed, put in tool plug conical flask, accurate addition 70% ethanol 50ml, close plug, weighed weight, supersound process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 70% ethanol, shake up, filter, take subsequent filtrate, to obtain final product；
Measure: precision draws reference substance solution and each 10 μ l of need testing solution respectively, inject chromatograph of liquid, measure, to obtain final product.
So far, complete the present invention, the invention has the beneficial effects as follows that have employed multiple method carries out comprehensive quality control to attached sweet medicine, it is ensured that the quality of medicine, strictly control the content of toxic component, it is ensured that active constituent content reaches requirement.
In order to further illustrate the present invention quality controllability for attached sweet medicine, further illustrate beneficial effects of the present invention below by way of quality standard research process of the present invention.Quality standard research is further intended to illustrate the effect of the present invention, and the restriction of non-invention.
One, sample preparation is measured
1, attached sweet granule is prepared
Method for making: take 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, and adds dextrin appropriate, mixing, wet granulation, dry, granulate, obtains the granule of attached sweet medicine.
According to above-mentioned preparation method, prepare four batch samples, lot number respectively 130901,140101,140102,140103.
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, quality standard research process
[character] is according to many batch samples observed result: this product is brown color extremely tan granule, the micro-hardship sweet, micro-of taste.
[discriminating]
1. Radix Aconiti Lateralis Preparata
1.1 discrimination methods
According to the method test under Radix Aconiti Lateralis Preparata assay item, in test sample chromatograph, the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time should be presented.
1.2 Method validation
See under sample [assay] Radix Aconiti Lateralis Preparata item.
The discriminating of Radix Aconiti Lateralis Preparata in 1.3 4 batch samples
Take four batch samples to check as a result, in four batches of test sample chromatographs, all present the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time.
2 Radix Glycyrrhizaes
2.1 discrimination methods
Employing thin layer chromatography differentiates.
Taking attached sweet granule 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 2 1) 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.
2.2, Method validation
2.2.1, specificity
Take attached sweet granule (130901 crowdes) 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 2 1) 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.
2.2.2, ruggedness
2.2.2.1, 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..
2.2.2.2, 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.
2.3 measure
Take three batch samples (140101 batches, 140102 batches, 140103 batches), differentiate Radix Glycyrrhizae according to preceding method as a result, in three batches of test sample chromatographs, the fluorescence speckle of all aobvious same color on position corresponding with control medicinal material and reference substance chromatograph.
[inspection]
1. diester-type alkaloids limit test
Diester-type alkaloids contained by Radix Aconiti Lateralis Preparata is the major toxicity composition of Radix Aconiti Lateralis Preparata medical material, for ensureing safety, tackle its limit to be controlled, testing with reference to diester-type alkaloids inspection method under " Chinese Pharmacopoeia " version (the second enlarged edition) mural nodules item in 2010, research process is as follows:
1.1 assay methods
The chromatographic column that chromatographic condition and system suitability are filler with octadecylsilane chemically bonded silica；With acetonitrile-oxolane (25 15) for mobile phase A, with 0.1% phosphoric acid solution containing 0.03mol/L potassium dihydrogen phosphate for Mobile phase B, the regulation according to the form below carries out gradient elution, and detection wavelength is 232nm.Number of theoretical plate calculates by aconitine peak should be not less than 30000.
Table 2 gradient elution program
It is appropriate that the preparation of reference substance solution takes mesaconitine, hypaconitine, aconitine reference substance, accurately weighed, add acetonitrile respectively and make every 1ml solution containing 0.2mg, as reference substance stock solution, precision measures each 5ml of reference substance stock solution, puts in 50ml measuring bottle, adds 0.1% phosphoric acid solution and is diluted to scale, shake up, to obtain final product.
nullSolid-phase extraction column system suitability precision measures each 3ml of reference substance stock solution，Mixing，Reduced pressure at room temperature is recycled to dry，Residue precision adds 0.1mol/L hydrochloric acid solution 50ml makes dissolving，Precision measures 5ml，It is placed in the solid-phase extraction column handled well and (exchanges reverse phase absorption agent for filler with mixed type cation，150mg，Capacity is 6ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with 0.lmol/L hydrochloric acid solution、Methanol、The each 5ml eluting of acetonitrile，Discard eluent，Place 5 minutes，Continue the mixed solution 10ml eluting with acetonitrile-strong ammonia solution (90 10)，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue precision adds the mixed solution 3ml of acetonitrile-0.1% phosphoric acid solution (30 70) 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 μ l of reference substance solution respectively, injects chromatograph of liquid, mensuration, computing system employment and suitability test (E & ST) solution and each corresponding Component peak area ratio of reference substance solution, cannot be less than 0.95.
The preparation of need testing solution takes finely ground rear this product content 1.0g, accurately weighed, put in tool plug conical flask, add the mixed solution 25ml of acetonitrile-strong ammonia solution (90 10), close plug, weighed weight, supersound process 30 minutes, filters, and filtrate is extremely dry in less than 40 DEG C decompression and solvent recoveries, residue precision adds 0.1mol/L hydrochloric acid solution 10ml makes dissolving, filtering, filtrate is according to the method under solid-phase extraction column system suitability item, from " being placed on the solid-phase extraction column handled well ", operate in accordance with the law, to obtain final product.
Algoscopy precision respectively draws reference substance solution and each 10 μ l of need testing solution, injects chromatograph of liquid, measures, to obtain final product.
1.2 Method validation
1.2.1 solid-phase extraction column system suitability experiment
Take mesaconitine, hypaconitine, aconitine reference substance, prepare reference substance solution according to said method；
Take reference substance stock solution, prepare solid-phase extraction column system suitability need testing solution according to said method.
Being measured according to above-mentioned liquid-phase condition and algoscopy as a result, solid-phase extraction column system suitability solution and each corresponding Component peak area ratio of reference substance solution are all higher than in 0.95, solid-phase extraction column system suitability is good.
1.2.2 specificity
Take 130901 batch samples and the negative preparation without Radix Aconiti Lateralis Preparata, prepare need testing solution according to said method；
It is measured according to above-mentioned liquid-phase condition and algoscopy as a result, test sample chromatograph is detecting (only detecting hypaconitine) to there being corresponding chromatographic peak on reference substance chromatograph relevant position, and separates well with adjacent chromatographic peak；Negative formulation samples chromatograph is not detecting chromatographic peak with reference substance chromatograph relevant position, it was shown that this law specificity is good.
1.2.3 limit is detected
Taking diester-type alkaloids reference substance solution stepwise dilution, sample size during with S/N for 31 is detection limit, tests, and result diester-type alkaloids detection limit is respectively as follows: aconitine 8ng, hypaconitine 8ng, mesaconitine 8ng.
1.2.4 ruggedness
1.2.4.1 stability of solution
Taking 130901 batch samples, prepare need testing solution, take need testing solution room temperature and place, drew 10 μ l respectively at the 0th, 6,12,18,24 hours, inject chromatograph of liquid, measure, result is in Table 3.
Table 3 solution stability testing result
As a result, need testing solution is stable in 24 hours.
1.2.4.2 mobile phase ratio
Taking sample, adopt different mobile phase ratio to be measured, result is in Table 4.
The different mobile phase ratio result of the test of table 4
It is shown that when mobile phase A changes in above-mentioned scope, in test sample chromatograph, parameters all meets the requirements, good tolerance.
1.2.4.3 chromatographic column
Taking sample, adopt the chromatographic column of different label to be measured, result is in Table 5.
The different chromatographic column result of the test of table 5
It is shown that this law chromatographic column good tolerance.
Diester-type alkaloids limit test in 1.3 3 batch samples
Taking three batch samples (140101 batches, 140102 batches, 140103 batches), check the limit of diester-type alkaloids in accordance with the law, result is in Table 6.
Table 6 three batch sample measurement result
Result shows, in three batch samples, the inspection result of diester-type alkaloids is between 23.9～25.6 μ g/ bags.
[assay]
1 Radix Aconiti Lateralis Preparata
With reference to " Chinese Pharmacopoeia " version (the second enlarged edition) mural nodules assay (Radix Aconiti Lateralis Preparata, Radix Aconiti Preparata) and " Chinese Pharmacopoeia " one high performance liquid chromatography of version in 2010 (annex VID) test in 2010.Measure the content of monoester alkaloid (Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine) in sample.
1.1 assay methods
Measure according to high performance liquid chromatography (one annex VI D of " Chinese Pharmacopoeia " version in 2010).
Chromatographic condition and system suitability: be connected the chromatographic column that phenyl bonded silica is filler with polarity ether；With the phosphoric acid solution (23 77) of acetonitrile-0.1% for mobile phase；Detection wavelength is 232nm.Number of theoretical plate calculates by Benzoylmesaconine peak and is 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%, shakes up, as solid-phase extraction column system suitability reference substance solution.Precision measures each reference substance stock solution 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 0.1%, 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.
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 not less than 0.95.
The preparation of need testing solution takes attached sweet granule, finely ground, weigh 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 0.1mol/L hydrochloric acid solution 25ml, close plug, weighed weight, supersound process 40 minutes jolting constantly, let cool, more weighed weight, the weight of less loss is supplied with 0.1mol/L hydrochloric acid solution, shake up, leaving the heart 30 minutes with per minute 4000, filter, precision measures subsequent filtrate 10ml, according to the method under solid-phase extraction column system suitability item, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, to obtain final product；
Algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, measures, to obtain final product.
1.2 CP method and method monoester alkaloid content of the present invention measure the suitability and compare
Radix Aconiti Lateralis Preparata monoester alkaloid content algoscopy (hereinafter referred to as " CP method ") and Radix Aconiti Lateralis Preparata monoester alkaloid content algoscopy of the present invention under one the second enlarged edition fugui gutong keli item of " Chinese Pharmacopoeia " version in 2010, the suitability measured for attached sweet granule Radix Aconiti Lateralis Preparata monoester alkaloid content is compared.
1.2.1 " CP method " measures attached sweet granule monoester alkaloid employment and suitability test (E & ST)
Chromatographic column that chromatographic condition and system suitability are filler with octadecylsilane chemically bonded silica (hereinafter referred to as: C18Post)；With acetonitrile-0.1% phosphoric acid solution (22:78) for mobile phase；Detection wavelength is 232nm.Number of theoretical plate calculates by Benzoylmesaconine peak and is all not less than 10000.
The preparation of reference substance solution: take Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance in right amount, accurately weighed, add acetonitrile respectively and make every 1ml solution containing 0.2mg, as reference substance stock solution；Precision measures each reference substance stock solution 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 0.1%, 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.
nullThe preparation of need testing solution: take attached sweet granule finely ground，Take 0.5g，Accurately weighed，Put in tool plug conical flask，Accurate addition 0.1mol/L hydrochloric acid solution 25ml，Close plug，Weighed weight，Supersound process 40 minutes jolting constantly，Let cool，Weighed weight again，The weight of less loss is supplied with 0.1mol/L hydrochloric acid solution，Shake up，Centrifugal (rotating speed is 5000 turns per minute) 30 minutes，Filter，Precision measures subsequent filtrate 10ml，It is added in solid-phase extraction column and (exchanges reverse phase absorption agent for filler with mixed type cation，150mg，Capacity is 6ml，Use acetonitrile in advance successively、The each 6ml eluting of water) on，Successively with water 3ml、Ammonia solution (5 → 100)、Water、Methanol、The each 5ml eluting of acetonitrile，After eluting liquid stream is most，Place 5 minutes，Continue the mixed solution 10ml eluting with acetonitrile-strong ammonia solution (90:10)，Collect eluent，In less than 40 DEG C decompression and solvent recoveries to dry，Residue precision adds the mixed solution 5ml of acetonitrile-0.1% phosphoric acid solution (20:80) makes dissolving，Filter，Take subsequent filtrate，Obtain.
Algoscopy: precision draws reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid, measures, to obtain final product.Determine the C of 3 different manufacturers18Post, need testing solution tailing factor result is in Table 7.
The different label C of table 718Post tailing factor measurement result
Result shows, when " CP method " measures this product monoester type alkaloid, and the C of 3 different labels18Post, Benzoylmesaconine, benzoyl hypo-aconine, benzoyl hypo-aconine chromatographic peak all trail seriously, and peak type is poor, affect the accurate measurement of peak area, therefore, C18Post is not suitable for the assay of attached sweet medicine monoester alkaloid.
1.2.2 the inventive method measures attached sweet granule monoester alkaloid employment and suitability test (E & ST)
According to above-mentioned " CP method " described experimental technique, liquid-phase condition, reference substance solution preparation method, need testing solution preparation method, algoscopy are all constant, simply chromatographic column is changed into and connect chromatographic column that phenyl bonded silica is filler (hereinafter referred to as phenyl post) with polarity ether, attached sweet granule monoester alkaloid is carried out assay.Determining the phenyl post of 3 different manufacturers, need testing solution tailing factor result is in Table 8:
The different label phenyl post tailing factor measurement result of table 8
Result shows, Benzoylmesaconine, benzoyl hypo-aconine, benzoyl hypo-aconine chromatographic peak tailing factor are all 0.95～1.05, chromatographic peak peak shape is good, and system suitability is good, it was shown that the inventive method is applicable to attached sweet granule monoester alkaloid content and measures.
1.3 CP method and method monoester type alkaloid content determination specificity of the present invention test are compared
1.3.1 " CP method " measures the test of attached sweet granule monoester alkaloid specificity
Take attached sweet granule and the negative granules agent without Radix Aconiti Lateralis Preparata, test according to " CP method " described liquid-phase condition and need testing solution preparation method.As a result, need testing solution chromatograph is having chromatographic peak with reference substance solution chromatograph corresponding position, but peak shape is poor, and hangover is serious, and Benzoylmesaconine chromatographic peak separating degree is less than 1.5；Negative sample solution is having chromatographic peak to detect with Benzoylmesaconine reference substance chromatograph corresponding position, it was shown that it is poor that " CP method " measures monoester alkaloid specificity in attached sweet granule.Detailed results is in Table 9.
Table 9 " CP method " specificity result
1.3.2 the inventive method measures the test of attached sweet granule monoester alkaloid specificity
Separately take attached sweet granule and the negative granules agent without Radix Aconiti Lateralis Preparata, test according to by liquid-phase condition described in the inventive method and need testing solution preparation method.Result, need testing solution chromatograph is having chromatographic peak with reference substance solution chromatograph corresponding position, and peak shape is good, and separating degree is good, negative sample solution is detecting without chromatographic peak with reference substance solution corresponding position, it was shown that it is good that the inventive method measures monoester alkaloid specificity in attached sweet granule.Detailed results is in Table 10.
Table 10 the inventive method specificity result
1.4 monoester alkaloid content mensuration methodology of the present invention researchs
1.4.1 solid-phase extraction column system suitability
Take Benzoylmesaconine, benzoyl aconine, benzoyl hypo-aconine reference substance, carry out solid-phase extraction column system suitability according to said method.
Result: solid-phase extraction column system suitability solution corresponding Component peak area ratio each to reference substance solution is all higher than 0.95, and detailed results is in Table 11.
Table 11 solid-phase extraction column system suitability result
1.4.2 accuracy experiment
The preparation of reference substance stock solution: take Benzoylmesaconine reference substance appropriate, accurately weighed, make every 1ml solution containing 3.7 μ g with 0.1mol/L hydrochloric acid solution, to obtain final product.
The preparation of need testing solution: take finely ground attached sweet granule 0.25g, accurately weighed 6 parts, precision adds above-mentioned reference substance stock solution 25ml respectively, according to the preparation method of need testing solution described in the inventive method, rise from " close plug, weighed weight ", operate in accordance with the law, obtain need testing solution.
Liquid-phase condition and algoscopy, with, described in the inventive method, measuring, calculate the response rate, and result is in Table 12.
Table 12 accuracy test result
As a result, accuracy is good.
1.4.3 precision
1.4.3.1 repeatability
Taking finely ground attached sweet granule 0.5g, accurately weighed 6 parts, measure according to the method for the invention, result is in Table 13.
Table 13 replica test result
It is shown that repeatability is good.
1.4.3.2 different instruments
On different instruments, same test sample being measured respectively, investigate the instrument variation impact on precision, result is in Table 14.
The different instrument test result of table 14
As a result, between different instruments, precision is good.
1.4.3.3 different tests personnel
By different personnel, same test sample being measured, investigate personnel's variation impact on precision, result is in Table 15.
The different personnel's result of the test of table 15
As a result, between different personnel, precision is good.
1.4.3.4 not same date
At not same date, same test sample being measured respectively by same analysis personnel, investigate the date variation impact on precision, result is in Table 16.
Table 16 different time result of the test
As a result, between same date, precision is not good.
1.4.4 ruggedness
1.4.4.1 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 is in Table 17.
Table 17 solution stability testing
As a result, need testing solution is stable in 12 hours.
1.4.4.2 wavelength is detected
Taking this product need testing solution, be measured respectively under 227nm, 232nm, 237nm wavelength, result is in Table 18.
Table 18 different wave length result of the test
As a result, wavelength good tolerance.
1.4.4.3 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 is in Table 19.
Table 19 different in flow rate result of the test
As a result, flow velocity good tolerance.
1.4.4.4 mobile phase ratio
Taking this product need testing solution, adopt different mobile phase ratio to be measured, result is in Table 20.
The different mobile phase ratio result of the test of table 20
As a result, mobile phase ratio good tolerance.
1.4.4.5 column temperature
Taking this product need testing solution, be respectively adopted 25 DEG C, 30 DEG C, 35 DEG C column temperatures and be measured, result is in Table 21.
The different column temperature result of the test of table 21
As a result, column temperature good tolerance.
1.4.4.6 chromatographic column label
Taking this product need testing solution, adopt the chromatographic column of different label to be measured, result is in Table 22.
The different chromatographic column result of the test of table 22
As a result, chromatographic column good tolerance.
1.4.5, linearly
1.4.5.1 Benzoylmesaconine
Take Benzoylmesaconine reference substance appropriate, accurately weighed, prepare every 1ml solution containing Benzoylmesaconine 3.3,9.8,16.3,22.9 and 32.7 μ g respectively with acetonitrile-0.1% phosphoric acid solution (20 80).Precision draws each 20 μ l of above-mentioned reference substance solution respectively, injects chromatograph of liquid, records chromatographic peak area.With Benzoylmesaconine reference substance solution concentration for abscissa, with corresponding peak area for vertical coordinate, carry out linear regression, obtain equation of linear regression: y=29732x-11339, r=0.9997.
As a result, Benzoylmesaconine is good linear relationship with peak area between 3.3～33.0 μ g/ml.
1.4.5.2 benzoyl aconine
Take benzoyl aconine reference substance appropriate, accurately weighed, prepare every 1ml solution containing benzoyl aconine 0.8,1.6,2.4,5.6 and 8.0 μ g respectively with acetonitrile-0.1% phosphoric acid solution (20 80).Precision draws each 20 μ l of above-mentioned reference substance solution respectively, injects chromatograph of liquid, records chromatographic peak area.With benzoyl aconine reference substance solution concentration for abscissa, with corresponding peak area for vertical coordinate, carry out linear regression, obtain equation of linear regression: y=245551x-2211.9, r=0.9999.
As a result, benzoyl aconine is good linear relationship with peak area between 0.8～8.0 μ g/ml.
1.4.5.3 benzoyl hypo-aconine
Take benzoyl hypo-aconine reference substance appropriate, accurately weighed, prepare every 1ml solution containing benzene first time aconine 0.8,1.6,2.4,4.0,5.6 and 8.0 μ g respectively with acetonitrile-0.1% phosphoric acid solution (20 80).Precision draws each 20 μ l of above-mentioned reference substance solution respectively, injects chromatograph of liquid, records chromatographic peak area.With benzoyl hypo-aconine reference substance solution concentration for abscissa, with corresponding peak area for vertical coordinate, carry out linear regression, obtain equation of linear regression: y=23916x-3973.3, r=0.9995.
As a result, benzoyl hypo-aconine is good linear relationship with peak area between 0.8～8.0 μ g/ml.
1.4.6 scope
Weighing this product content by the 80% of 0.5g, 100%, 120% respectively, prepare need testing solution, measure monoester alkaloid content, result is in Table 23.
Table 23 scope result of the test
As a result, within the scope of the sampling of 0.5g ± 20%, do not find that measurement result has notable difference.
The assay of monoester alkaloid in 1.5 3 batch samples
Taking three batches of quick granules (140101 batches, 140102 batches, 140103 batches), measure monoester alkaloid content, result is in Table 24.
Table 24 3 batch sample assay result
2 Radix Glycyrrhizaes
With reference to " Chinese Pharmacopoeia 2005 version () and the method under " Chinese Pharmacopoeia " version (one) licorice medicinal materials assay item in 2010 are tested, and measure the content of liquirtin in developing products.
2.1 methods
Chromatographic condition and system suitability: the chromatographic column being filler with octadecylsilane chemically bonded silica；With acetonitrile-0.5% glacial acetic acid (18 82) for mobile phase；Detection wavelength is 276nm；Number of theoretical plate calculates by liquirtin peak should be not less than 4000.
Reference substance solution to prepare extracting liquorice glycosides reference substance appropriate, accurately weighed, add 70% ethanol and make every 1ml solution containing 20 μ g, to obtain final product.
It is finely ground that the preparation of need testing solution takes attached sweet granule, takes 0.5g, accurately weighed, puts in tool plug conical flask, accurate addition 70% ethanol 50ml, close plug, weighed weight, supersound process 30 minutes, let cool, more weighed weight, supply the weight of less loss with 70% ethanol, shake up, filter, take subsequent filtrate, to obtain final product.
Algoscopy precision respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injects chromatograph of liquid, measures, to obtain final product.
2.2 Method validation
2.2.1 specificity
The preparation of need testing solution takes 130901 batches quick granules and the negative preparations without Radix Glycyrrhizae, prepares need testing solution according to said method.
Be measured according to above-mentioned liquid-phase condition and algoscopy as a result, in test sample chromatograph with reference substance chromatograph relevant position on have the chromatographic peak of identical retention time, separating degree is good, negative sample solution detects without chromatographic peak in relevant position, it was shown that negative noiseless, and it is good that this law measures Radix Glycyrrhizae specificity.
2.2.2 accuracy
Accuracy test contrast solution to prepare extracting liquorice glycosides reference substance appropriate, accurately weighed, add 70% ethanol and make every 1ml reference substance solution containing 0.1mg.
It is finely ground that the preparation of accuracy test need testing solution takes 130901 batches of contents, take 0.25g, accurately weighed, totally 6 parts, precision adds above-mentioned liquirtin reference substance solution (0.1mg/ml) 5ml respectively, according to preferred need testing solution preparation method, from " putting in tool plug conical flask ", operate in accordance with the law, to obtain final product.
Table 25 accuracy test result
As a result, accuracy is good.
2.2.3 precision
2.2.3.1 repeatability
Take 130901 batches of contents finely ground, take 0.5g, accurately weighed, totally 6 parts, operate by preferred need testing solution preparation method, measure liquirtin content respectively.Result is in Table 26.
Table 26 replica test result
As a result, repeatability is good.
2.2.3.2 different personnel
Taking 130901 batches quick granules, measured liquirtin content by different personnel respectively, investigate personnel's variation impact on precision, result is in Table 27.
The different personnel's result of the test of table 27
As a result, the precision of different personnel's tests is good.
2.2.3.4 different instruments
Taking 130901 batches quick granules, measure liquirtin content respectively on different instruments, investigate the instrument variation impact on precision, result is in Table 28.
The different instrument test result of table 28
As a result, the precision of different instrument tests is good.
2.2.3.5 different time
Take 130901 batches quick granules, measure liquirtin content at not same date respectively, investigate the date variation impact on precision.Result is in Table 29.
Table 29 different time result of the test
As a result, the precision of different time test is good.
2.2.4 linear
Extracting liquorice glycosides reference substance is appropriate, and being respectively prepared concentration with 70% ethanol is every 1ml reference substance solution containing liquirtin 8,16,32,48,64 μ g；Precision draws each 10 μ l of above-mentioned reference substance solution respectively, injects chromatograph of liquid, records chromatogram.With liquirtin reference substance solution concentration for abscissa, with peak area for vertical coordinate, carrying out linear regression, obtaining regression equation is: y=19203x+2039.4, r=0.9997.
It is shown that liquirtin is good linear relationship with peak area between 8～64 μ g/ml.
2.2.5 ruggedness
2.2.5.1 stability of solution
Taking 130901 batches quick granules and prepare need testing solution, room temperature is placed, respectively at the 0th, 5,10,15,20 hours, and the accurate 10 μ l that draw, injection chromatograph of liquid, record liquirtin chromatographic peak area, result is in Table 30.
Table 30 solution stability testing result
As a result, need testing solution is stable in 20 hours.
2.2.5.2 column temperature
Taking 130901 batches quick granules and prepare need testing solution, be respectively adopted 25 DEG C, 30 DEG C, 35 DEG C column temperature analyses, measure liquirtin content, result is in Table 31.
The different column temperature result of the test of table 31
As a result, the good tolerance of different column temperature tests.
2.2.5.3 flow velocity
Take 130901 batches quick capsules and prepare need testing solution, be respectively adopted 0.8ml/min, 1.0ml/min, 1.2ml/min flow velocity analysis, measure liquirtin content.
Table 32 different in flow rate result of the test
As a result, the good tolerance of mobile phase different in flow rate test.
2.2.5.4 wavelength is detected
Taking 130901 batches quick granules and prepare need testing solution, analyze respectively, measure liquirtin content under 227nm, 232nm, 237nm wavelength, result is in Table 33.
Table 33 different wave length result of the test
As a result, the good tolerance of different detection wavelength tests.
2.2.5.5 mobile phase ratio
Taking 130901 batches quick granules and prepare need testing solution, be respectively adopted different mobile phase ratio analysis, measure liquirtin content, result is in Table 34.
The different mobile phase ratio result of the test of table 34
As a result, the good tolerance of different mobile phase ratio tests.
2.2.5.6 chromatographic column
Taking 130901 batches quick granules and prepare need testing solution, adopt the chromatogram column analysis of different label, measure liquirtin content, result is in Table 35.
The different chromatographic column result of the test of table 35
As a result, the good tolerance of different chromatographic column tests.
2.2.6 scope
Taking 130901 batches quick granular contents finely ground, press the 50% of sampling amount 0.5g, 100%, 150% accurate title respectively and measure liquirtin content, result is in Table 36.
Table 36 scope result of the test
As a result, within the scope of the sampling of 0.5g ± 0.25g, do not find that measurement result has notable difference.
The assay of liquirtin in 2.3 3 batch samples
Taking three batch samples (140101 batches, 140102 batches, 140103 batches), measure liquirtin content in accordance with the law, result is in Table 37.
The result of liquirtin assay in table 37 3 batch sample
As a result, in three batch samples the content of liquirtin between 3.22～3.63mg/ grain.
Detailed description of the invention
Embodiment 1: attached sweet granule monoester alkaloid content measures
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) inspection
Taking sample, test according to quality standard of the present invention, assay is shown in following table:
Table 38 assay
Embodiment 2: attached sweet granule monoester alkaloid content measures
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) inspection
Taking sample, test according to quality standard of the present invention, assay is shown in following table:
Table 39 assay
Embodiment 3: attached sweet granule monoester alkaloid content measures
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) inspection
Taking sample, test according to quality standard of the present invention, assay is shown in following table:
Table 40 assay
Embodiment 4: attached sweet tablet monoester alkaloid content measures
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, taking filtrate reduced in volume to relative density is 1.12 (60 DEG C), adds ethanol, just adds just stirring and evenly mixing, alcohol content is made to reach 70%, stand 24h, filter, filtrate recycling ethanol, being evaporated to relative density is 1.25 (60 DEG C), less than 60 DEG C drying under reduced pressure, pulverize, and add starch appropriate, granulate, add Pulvis Talci appropriate, mixing, tabletting, sugar coating, obtains the tablet of attached sweet medicine.
2) inspection
Taking sample, test according to quality standard of the present invention, assay is shown in following table:
Table 41 assay
Embodiment 5: attached sweet capsule monoester alkaloid content measures
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, taking filtrate reduced in volume to relative density is 1.15 (60 DEG C), add ethanol, just just stirring and evenly mixing is added, alcohol content is made to reach 80%, stand 12h, filter, filtrate recycling ethanol, being evaporated to relative density 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) inspection:
Taking sample, test according to quality standard of the present invention, assay is shown in following table:
Table 42 assay
Claims (1)
1. an attached sweet drug detection method, it is characterised in that: differentiate Radix Aconiti Lateralis Preparata by high performance liquid chromatography, differentiate Radix Glycyrrhizae by thin layer chromatography, by high effective liquid chromatography for measuring diester-type alkaloids limit, with high effective liquid chromatography for measuring Radix Aconiti Lateralis Preparata and Radix Glycyrrhizae content；
[character] this product content be brown color to sepia, the micro-hardship sweet, micro-of taste；
[discriminating] (1) Radix Aconiti Lateralis Preparata differentiates: according to the method test under Radix Aconiti Lateralis Preparata assay item, test sample chromatograph should present the chromatographic peak corresponding with Benzoylmesaconine, benzoyl aconine and benzoyl hypo-aconine reference substance chromatographic peak retention time, namely determine it is the preparation containing Radix Aconiti Lateralis Preparata；
(2) Radix Glycyrrhizae differentiates: taking this product content 0.5-2.5g, 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, wash with water 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 VI B thin layer chromatography test of " Chinese Pharmacopoeia " version in 2010, draw each 5 μ l of above-mentioned three 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；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；
[inspection] diester-type alkaloids: according to one annex VI D high effective liquid chromatography for measuring of " Chinese Pharmacopoeia " version in 2010；
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica for filler；With acetonitrile tetrahydrofuran=25 15 for mobile phase A, with 0.1% phosphoric acid solution containing 0.03mol/L potassium dihydrogen phosphate for Mobile phase B, carrying out gradient elution by following regulation, detection wavelength is 232nm；Number of theoretical plate calculates by aconitine peak should be not less than 30000；
Gradient elution program: at 0～38 minute, the ratio of mobile phase A was increased to 26% by 15%, and the ratio of Mobile phase B is reduced to 74% by 85%；At 38～39 minutes, the ratio of mobile phase A was increased to 35% by 26%, and the ratio of Mobile phase B is reduced to 65% by 74%；At 39～49 minutes, the ratio of mobile phase A was 35%, and the ratio of Mobile phase B is 65%；
It is appropriate that the preparation of reference substance solution takes mesaconitine reference substance, hypaconitine reference substance, aconitine reference substance, accurately weighed, add acetonitrile respectively and make every 1ml solution containing 0.2mg, as reference substance stock solution, precision measures each 5ml of reference substance stock solution, puts in 50ml measuring bottle, adds 0.1% phosphoric acid solution and is diluted to scale, shake up, to obtain final product；
Solid-phase extraction column system suitability precision measures each 3ml of reference substance stock solution, mixing, reduced pressure at room temperature is recycled to dry, residue precision adds 0.1mol/L hydrochloric acid solution 50ml makes dissolving, precision measures 5ml, it is placed on the solid-phase extraction column handled well, successively with 0.lmol/L hydrochloric acid solution, methanol, the each 5ml eluting of acetonitrile, discard eluent, place 5 minutes, continue 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 precision adds the mixed solution 3ml of acetonitrile 0.1% phosphoric acid solution=30 70 makes dissolving, filter, take subsequent filtrate as solid-phase extraction column system suitability solution；Described solid-phase extraction column exchanges reverse phase absorption agent for filler with mixed type cation, 150mg, and capacity is 6ml, in advance successively with acetonitrile, each 6ml eluting of water；
Precision draws above-mentioned solid phase extraction column system suitability solution and each 10 μ l of reference substance solution respectively, injects chromatograph of liquid, mensuration, computing system employment and suitability test (E & ST) solution and each corresponding Component peak area ratio of reference substance solution, cannot be less than 0.95；
The preparation of need testing solution: take this product appropriate, finely ground, take 1～3g, accurately weighed, put in tool plug conical flask, the accurate mixed solution 50ml adding acetonitrile strong ammonia solution=90 10, close plug, weighed weight, supersound process 30 minutes, the weight of less loss is supplied with the mixed solution of acetonitrile strong ammonia solution=90 10, shake up, filter, precision measures 25ml subsequent filtrate in less than 40 DEG C decompression and solvent recoveries to dry, residue precision adds 0.1mol/L hydrochloric acid solution 10ml makes dissolving, filter, filtrate is according to the method under solid-phase extraction column system suitability item, from " being placed on the solid-phase extraction column handled well ", operate in accordance with the law, obtain；
Algoscopy: precision draws reference substance solution and each 10 μ l of need testing solution respectively, injects chromatograph of liquid, measures, to obtain final product；
Other should meet every regulation relevant under one annex rules of preparations item of " Chinese Pharmacopoeia " version in 2010；
[assay] Radix Aconiti Lateralis Preparata assay: according to one annex VI D high effective liquid chromatography for measuring of " Chinese Pharmacopoeia " version in 2010；
Chromatographic condition and system suitability: be connected the chromatographic column that phenyl bonded silica is filler with polarity ether；With the phosphoric acid solution of acetonitrile 0.1%=23 77 for mobile phase；Detection wavelength is 232nm, and number of theoretical plate calculates by Benzoylmesaconine peak and is 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, put in the measuring bottle of same 100ml, it is diluted to scale with the phosphoric acid solution of 0.1%, shake up, as solid-phase extraction column system suitability reference substance solution, precision measures each reference substance stock solution 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 0.1%, 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；
Solid-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 on the solid-phase extraction column handled well, 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；Described solid-phase extraction column exchanges reverse phase absorption agent for filler with mixed type cation, and specification is 150mg, 6ml, in advance successively with acetonitrile, each 6ml eluting of water；
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 not less than 0.95；
It is appropriate that the preparation of need testing solution takes this product, finely ground, weigh 0.2～0.6g, accurately weighed, put in tool plug conical flask, accurate addition 0.1mol/L hydrochloric acid solution 25ml, close plug, weighed weight, supersound process 40 minutes jolting constantly, let cool, more weighed weight, the weight of less loss is supplied with 0.1mol/L hydrochloric acid solution, shake up, leaving the heart 30 minutes with per minute 4000, filter, precision measures subsequent filtrate 10ml, according to the method under solid-phase extraction column system suitability item, from " being placed in the solid-phase extraction column handled well ", operate in accordance with the law, to obtain final product；
Algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, measures, to obtain final product；
Radix Glycyrrhizae assay: according to one annex VI D high effective liquid chromatography for measuring of " Chinese Pharmacopoeia " version in 2010；
Chromatographic condition and system suitability: the chromatographic column being filler with octadecylsilane chemically bonded silica；With acetonitrile 0.5% glacial acetic acid solution=18 82 for mobile phase；Detection wavelength is 276nm；Number of theoretical plate calculates by liquirtin peak should be not less than 4000；
The preparation of reference substance solution: extracting liquorice glycosides reference substance is appropriate, accurately weighed, adds 70% ethanol and makes every 1ml solution containing 20 μ g, to obtain final product；
The preparation of need testing solution: take this product content, finely ground, take 0.2～0.6g, accurately weighed, put in tool plug conical flask, accurate addition 70% ethanol 50ml, close plug, weighed weight, supersound process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 70% ethanol, shake up, filter, take subsequent filtrate, to obtain final product；
Measure: precision draws reference substance solution and each 10 μ l of need testing solution respectively, inject chromatograph of liquid, measure, to obtain final product.
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