CN104833754A - Method for quality detection of monkshood-radix glycyrrhizae medicament - Google Patents
Method for quality detection of monkshood-radix glycyrrhizae medicament Download PDFInfo
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Abstract
The invention provides a method for the quality detection of a monkshood-radix glycyrrhizae medicament. The method is characterized in that a chromatographic column taking a polar diethyl ether connected phenyl bonded silica gel as a filler is adopted for identifying and determining the content of monkshood, so that the separating degree, peak shape and tailing factor of a target component are significantly improved; a thin-layer chromatography is adopted for identifying radix glycyrrhizae; high performance liquid chromatography is adopted for controlling the content of diester-type alkaloids; and related methodology validation and example inspection show that according to the method provided by the invention, the monkshood-radix glycyrrhizae medicament can be simply and accurately identified, the content of diester-type alkaloids can be controlled, and the contents of monkshood and radix glycyrrhizae can be detected, so that the product quality can be effectively guaranteed in the processes of production and use, and then the curative effect of the medicament is ensured.
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
The medicine of Chinese invention patent (ZL201210536161.3) a kind for the treatment of of allergic rhinitis and bronchial astehma, by monkshood 6-18 part, Radix Glycyrrhizae 9-1 part is that raw material is made (called after " attached sweet medicine "), has good curative effect for allergic rhinitis and bronchial astehma.
Allergic rhinitis and bronchial astehma are the closely-related respiratory disease of type Ⅰ allergy.Belong to frequently-occurring disease, difficult disease, and morbidity rate there is the trend increased year by year.The medicine of existing treatment of allergic rhinitis mainly contains antihistaminic, glucocorticoids, anti-leukotriene medicine, anticholinergic drug and adrenergic etc., mainly nasal-cavity administration, Long-Time Service easily has side effects and drug resistance, and recurrence rate after healing is high, and long-term efficacy is poor.In addition desensitizing immunization therapy can not large area clinical expansion because the reasons such as the course for the treatment of is long are restricted.The modified form of the at present both at home and abroad research of this type of medicine mainly existing medicine, emphasis is the long-acting dosage form of spray, inhalant, to reduce times for spraying, reduces spinoff, improves the compliance of patient.The feature of above-mentioned chemical drug is rapid-action, and effect is obvious, but long-term effect is poor, can not effect a radical cure, and easily produces drug resistance and spinoff.
The existing clinical practice result of the present invention shows, this product is good to the determined curative effect of allergic rhinitis and bronchial astehma, long-term effect, have no bad reaction, compensate for the defect that chemical drug only controls symptom, long-term effect difference etc. to a certain extent.
The prescription of attached sweet medicine contains monkshood, and alkaloid contained by monkshood mainly contains 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 monkshood, hypertoxic composition is that diester-type alkaloids is mainly aconitine, mesaconine and Hypaconitine.Oral di-esters alkaloid 3 ~ 4mg can the causing death (LD of studies on acute toxicity
50for 11.8mg/Kg), illustrate that di-esters alkaloid toxicity is extremely strong, it is the limited main cause of clinical practice.
Attached sweet medicine of the present invention, prior art does not have the quality determining method of preparation, effectively cannot ensure product quality, therefore in production with in using, in order to ensure attached sweet drug quality and clinical application effect and safety, provide a set of easy, reliable quality determining method extremely important.
Summary of the invention
The object of this invention is to provide a kind of quality determining method of attached sweet medicine, define the limit of effective component content and toxic component (dibasic acid esters alkaloid), to ensure Drug safety, validity and quality controllability.
Object 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, is characterized in that:
[proterties]this product content be brown color to sepia, micro-sweet, the micro-hardship of taste;
[discriminating](1) monkshood is differentiated: according to the method test under monkshood assay item, should present the chromatographic peak corresponding with benzoylmesaconine, benzoyl aconine and benzoyl time aconine reference substance chromatographic peak retention time in test sample chromatogram;
(2) Radix Glycyrrhizae is differentiated: get this product content 0.5 ~ 2.5g, add diethyl ether 40ml, adds hot reflux 1 hour, filter, discard ether liquid, the dregs of a decoction add methyl alcohol 30ml, add hot reflux 1 hour, filter, filtrate evaporate to dryness, the residue 40ml that adds water makes dissolving, extracts 3 times, each 20ml with normal butyl alcohol, merge normal butyl alcohol liquid, wash 3 times with water, discard water liquid, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Another extracting Radix Glycyrrhizae control medicinal material 1g, is made in the same way of control medicinal material solution; Extracting Radix Glycyrrhizae glycosides reference substance again, adds methyl alcohol and makes the solution of every 1ml containing 2mg, product solution in contrast; Test according to thin-layered chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw each 5 μ l of above-mentioned three kinds of solution, put on the silica gel g thin-layer plate prepared in same use 1% sodium hydroxide solution respectively, with normal butyl alcohol-strong ammonia solution-ethanol (5 ﹕ 2 ﹕ 1) for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear, inspects under putting ultraviolet lamp (365nm); In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material, the fluorescence spot of aobvious same color;
[inspection]diester-type alkaloids: measure according to high performance liquid chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI D);
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; 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, the regulation according to the form below carries out gradient elution, and determined wavelength is 232nm; Number of theoretical plate calculates should be not less than 30000 by aconitine peak;
Table 1 gradient elution program
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0~38 | 15→26 | 85→74 |
| 38~39 | 26→35 | 74→65 |
| 39~49 | 35 | 65 |
The preparation of reference substance solution gets mesaconine reference substance, Hypaconitine reference substance, aconitine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes the solution of every 1ml containing 0.2mg, product stock solution in contrast; 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, be placed in the solid-phase extraction column handled well (with the agent of mixed type cation exchange reverse phase absorption for filling agent, 150mg, capacity is 6ml, use acetonitrile successively in advance, the each 6ml wash-out of water) on, successively with 0.lmol/L hydrochloric acid solution, methyl alcohol, the each 5ml wash-out of acetonitrile, discard eluent, place 5 minutes, continue with the mixed solution 10ml wash-out of acetonitrile-strong ammonia solution (90 ﹕ 10), collect eluent, in less than 40 DEG C decompression and solvent recoveries to dry, the mixed solution 3ml that residue precision adds acetonitrile-0.1% phosphoric acid solution (30 ﹕ 70) makes dissolving, filter, get subsequent filtrate as solid-phase extraction column system suitability solution,
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 10 μ l of reference substance solution respectively, injection liquid chromatography, measure, computing system employment and suitability test (E & ST) solution and each corresponding Component peak area ratio of reference substance solution, must not be less than 0.95;
The preparation of need testing solution: it is appropriate to get this product, porphyrize, get 1 ~ 3g, accurately weighed, put in tool plug conical flask, precision adds the mixed solution 50ml of acetonitrile-strong ammonia solution (90 ﹕ 10), close plug, weighed weight, ultrasonic 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 " be placed in handle well solid-phase extraction column on ", operate in accordance with the law, obtain,
Determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product;
otherevery regulation relevant under should meeting " Chinese Pharmacopoeia " version in 2010 annex rules of preparations item;
[assay]monkshood assay: measure according to high performance liquid chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI D);
Chromatographic condition and system suitability: be connected the chromatographic column that phenyl bonded silica is filling agent with polarity ether; With the phosphoric acid solution of acetonitrile-0.1% (23 ﹕ 77) for mobile phase; Determined wavelength is 232nm, and number of theoretical plate calculates by benzoylmesaconine peak and is not less than 5000;
The preparation of reference substance solution gets benzoylmesaconine, benzoyl aconine, benzoyl time aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes the solution of every 1ml containing 0.2mg, product stock solution in contrast; Precision measures above-mentioned reference substance stock solution each 10ml, 5ml and 5ml respectively, put in the measuring bottle of same 100ml, scale is diluted to 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, scale is diluted to the phosphoric acid solution of 0.1%, shake up, be prepared into the reference substance solution of every 1ml containing benzoylmesaconine 15 μ g, benzoyl aconine 2 μ g, benzoyl time aconine 3 μ g;
Solid-phase extraction column system suitability: precision measures benzoylmesaconine, benzoyl aconine, the each 10ml of reference substance stock solution of benzoyl time 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, be placed in the solid-phase extraction column handled well (with the agent of mixed type cation exchange reverse phase absorption for filling agent, 150mg, 6ml, use acetonitrile successively in advance, the each 6ml wash-out of water) on, successively with water 3ml, the ammonia solution of 1.25%, water, methyl alcohol, the each 5ml wash-out of acetonitrile, after eluent flows to end, place 5 minutes, use the mixed solution 10ml wash-out of acetonitrile-strong ammonia solution (90 ﹕ 10) again, collect eluent, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds mobile phase 5ml makes dissolving, filter, get subsequent filtrate as solid-phase extraction column system suitability solution,
Accurate absorption said system employment and suitability test (E & ST) solution and each 20 μ l of reference substance solution respectively, injection liquid chromatography, measure, computing system employment and suitability test (E & ST) solution and the ratio of corresponding Component peak area each in reference substance solution, be all not less than 0.95;
Attached sweet particle is got in the preparation of need testing solution, porphyrize, takes 0.5g, accurately weighed, put in tool plug conical flask, precision adds 0.1mol/L hydrochloric acid solution 25ml, close plug, weighed weight, ultrasonic process also jolting constantly in 40 minutes, let cool, more weighed weight, the weight of less loss is supplied with 0.1mol/L hydrochloric acid solution, shake up, leave 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 ", operating in accordance with the law, to obtain final product;
Determination method is accurate respectively draws reference substance solution and each 20 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
Radix Glycyrrhizae assay: measure according to high performance liquid chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI D);
Chromatographic condition and system suitability: the chromatographic column taking octadecylsilane chemically bonded silica as filling agent; With acetonitrile-0.5% glacial acetic acid solution (18 ﹕ 82) for mobile phase; Determined wavelength is 276nm; Number of theoretical plate calculates should be not less than 4000 by liquiritin peak;
The preparation of reference substance solution: extracting Radix Glycyrrhizae glycosides reference substance is appropriate, accurately weighed, adds 70% ethanol and makes the solution of every 1ml containing 20 μ g, to obtain final product;
The preparation of need testing solution: get this product content, porphyrize, get 0.2 ~ 0.6g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 70% ethanol, shake up, filter, get subsequent filtrate, to obtain final product;
Measure: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, 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, can ensure the quality of medicine, strictly control the content of toxic component, ensure effective component content and reach requirement.
In order to further illustrate the quality controllability of the present invention for attached sweet medicine, further illustrate beneficial effect of the present invention below by way of quality standard research process of the present invention.Quality standard research is intended to further illustrate effect of the present invention, but not restriction of the present invention.
One, working sample preparation
1, attached sweet granule is prepared
Method for making: get monkshood 15kg, Radix Glycyrrhizae 6kg, boiling twice, first time adds 8 times of water gagings, decocts 2 hours, and second time adds 6 times of water gagings, decoct 1 hour, merge twice decocting liquid, filter, concentrated, drying under reduced pressure, pulverizes, and it is appropriate to add dextrin, mixing, wet granulation, dry, whole grain, obtains the granule of attached sweet medicine.
According to above-mentioned preparation method, prepare four batch samples, lot number is respectively 130901,140101,140102,140103.
2, preparation is not containing the negative granules agent of monkshood
Extracting Radix Glycyrrhizae 300g, the method according to the attached sweet granule of preparation is made not containing the negative granules agent of monkshood.
3, preparation is not containing the negative granules agent of Radix Glycyrrhizae
Get monkshood 750g, the method according to the attached sweet granule of preparation is made not containing the negative granules agent of Radix Glycyrrhizae.
Two, quality standard research process
[proterties]according to many batch samples observations: this product is brown color extremely tan particle, micro-sweet, the micro-hardship of taste.
[discriminating]
1. monkshood
1.1 discrimination method
According to the method test under monkshood assay item, in test sample chromatogram, the chromatographic peak corresponding with benzoylmesaconine, benzoyl aconine and benzoyl time aconine reference substance chromatographic peak retention time should be presented.
1.2 Method validation
Under seeing sample [assay] monkshood item.
the discriminating of monkshood in 1.3 4 batch samples
Get four batch sample inspections, result, in four batches of test sample chromatograms, all present the chromatographic peak corresponding with benzoylmesaconine, benzoyl aconine and benzoyl time aconine reference substance chromatographic peak retention time.
2 Radix Glycyrrhizaes
2.1 discrimination method
Employing thin-layered chromatography is differentiated.
Get attached sweet particle porphyrize, take 2g, add diethyl ether 40ml, adds hot reflux 1 hour, filter, discard ether liquid, the dregs of a decoction add methyl alcohol 30ml, add hot reflux 1 hour, filter, filtrate evaporate to dryness, the residue 40ml that adds water makes dissolving, extracts 3 times, each 20ml with normal butyl alcohol, merge normal butyl alcohol liquid, with equal-volume water washing 3 times, discard water liquid, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Another extracting Radix Glycyrrhizae control medicinal material 1g, is made in the same way of control medicinal material solution.Extracting Radix Glycyrrhizae glycosides reference substance again, adds methyl alcohol and makes the solution of every 1ml containing 2mg, product solution in contrast.Draw each 5 μ l of above-mentioned four kinds of solution, put on the silica gel g thin-layer plate prepared in same use 1% sodium hydroxide solution respectively, with normal butyl alcohol-strong ammonia solution-ethanol (5 ﹕ 2 ﹕ 1) for developping agent, launch, take out, dry, spray with 10% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting 365nm ultraviolet lamp.In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material, the fluorescence spot of aobvious same color, namely determines it is the preparation containing Radix Glycyrrhizae.
2.2, Method validation
2.2.1, specificity
Get attached sweet particle (130901 crowdes) 2g, prepare need testing solution according to said method.
Extracting Radix Glycyrrhizae control medicinal material 1g, is made in the same way of control medicinal material solution.
Extracting Radix Glycyrrhizae glycosides reference substance, adds methyl alcohol and makes the solution of every 1ml containing 2mg, product solution in contrast.
Get not containing the negative preparation 2g of Radix Glycyrrhizae, prepare negative need testing solution according to need testing solution preparation method.
Draw each 5 μ l of above-mentioned four kinds of solution, put on the silica gel g thin-layer plate prepared in same use 1% sodium hydroxide solution respectively, with normal butyl alcohol-strong ammonia solution-ethanol (5 ﹕ 2 ﹕ 1) for developping agent, launch, take out, dry, spray with 10% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting 365nm ultraviolet lamp.
As a result, in test sample chromatogram, the fluorescence spot of aobvious same color on the position corresponding with liquiritin reference substance chromatogram to Radix Glycyrrhizae control medicinal material; Negative sample solution is at relevant position immaculate.Illustrate that this law differentiates that Radix Glycyrrhizae specificity is good.
2.2.2, durability
2.2.2.1, stability of solution
Get need testing solution, control medicinal material solution, reference substance solution and developping agent under specificity item, ambient temperatare is put, respectively at the 0th, 4,8 hour point sample, expansion.It is good that the above-mentioned each solution of result display places 8 hours internal stabilities in room temperature, can effectively differentiate Radix Glycyrrhizae.。
2.2.2.2, launch temperature
Get 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 the method and launches well, can effectively differentiate Radix Glycyrrhizae within the scope of said temperature.
2.3 measure
Get three batch samples (140101 batches, 140102 batches, 140103 batches), differentiate Radix Glycyrrhizae according to preceding method, result, in three batches of test sample chromatograms, the fluorescence spot of all aobvious same color on the position corresponding with reference substance chromatogram to control medicinal material.
[inspection]
1. diester-type alkaloids limit test
Diester-type alkaloids contained by monkshood is the major toxicity composition of monkshood medicinal material, for ensureing security, tackle its limit to control, test 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 method
Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as the chromatographic column of filling agent; 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, the regulation according to the form below carries out gradient elution, and determined wavelength is 232nm.Number of theoretical plate calculates should be not less than 30000 by aconitine peak.
Table 2 gradient elution program
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0~38 | 15→26 | 85→74 |
| 38~39 | 26→35 | 74→65 |
| 39~49 | 35 | 65 |
Mesaconine is got in the preparation of reference substance solution, Hypaconitine, aconitine reference substance are appropriate, accurately weighed, add acetonitrile respectively and make the solution of every 1ml containing 0.2mg, product stock solution in contrast, 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, be placed in the solid-phase extraction column handled well (with the agent of mixed type cation exchange reverse phase absorption for filling agent, 150mg, capacity is 6ml, use acetonitrile successively in advance, the each 6ml wash-out of water) on, successively with 0.lmol/L hydrochloric acid solution, methyl alcohol, the each 5ml wash-out of acetonitrile, discard eluent, place 5 minutes, continue with the mixed solution 10ml wash-out of acetonitrile-strong ammonia solution (90 ﹕ 10), collect eluent, in less than 40 DEG C decompression and solvent recoveries to dry, the mixed solution 3ml that residue precision adds acetonitrile-0.1% phosphoric acid solution (30 ﹕ 70) makes dissolving, filter, get subsequent filtrate as solid-phase extraction column system suitability solution.
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 10 μ l of reference substance solution respectively, injection liquid chromatography, measure, computing system employment and suitability test (E & ST) solution and each corresponding Component peak area ratio of reference substance solution, must not be less than 0.95.
This product content 1.0g after porphyrize is got in the preparation of need testing solution, accurately weighed, put in tool plug conical flask, add the mixed solution 25ml of acetonitrile-strong ammonia solution (90 ﹕ 10), close plug, weighed weight, ultrasonic process 30 minutes, filters, and filtrate is 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 according to the method under solid-phase extraction column system suitability item, from " be placed in handle well solid-phase extraction column on ", operate in accordance with the law, to obtain final product.
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
1.2 Method validation
1.2.1 solid-phase extraction column system suitability is tested
Get mesaconine, Hypaconitine, aconitine reference substance, prepare reference substance solution according to said method;
Get reference substance stock solution, prepare solid-phase extraction column system suitability need testing solution according to said method.
Measure according to above-mentioned liquid-phase condition and determination method, result, solid-phase extraction column system suitability solution and each corresponding Component peak area ratio of reference substance solution are all greater than in 0.95, and solid-phase extraction column system suitability is good.
1.2.2 specificity
Get 130901 batch samples and not containing the negative preparation of monkshood, prepare need testing solution according to said method;
Measure according to above-mentioned liquid-phase condition and determination method, result, test sample chromatogram is having corresponding chromatographic peak to detect (only detecting Hypaconitine) on reference substance chromatogram relevant position, and is separated well with adjacent chromatographic peak; Negative formulation samples chromatogram, not detecting chromatographic peak with reference substance chromatogram relevant position, shows that this law specificity is good.
1.2.3 detectability
Get diester-type alkaloids reference substance solution, and stepwise dilution, sample size when being 3 ﹕ 1 with S/N is detectability, tests, and result diester-type alkaloids detectability is respectively: aconitine 8ng, Hypaconitine 8ng, mesaconine 8ng.
1.2.4 durability
1.2.4.1 stability of solution
Get 130901 batch samples, prepare need testing solution, get need testing solution room temperature and place, drew 10 μ l respectively at the 0th, 6,12,18,24 hour, injection liquid chromatography, measure, the results are shown in Table 3.
Table 3 solution stability testing result
| Standing time (hr) | 0 | 6 | 12 | 18 | 24 | RSD |
| Peak area | 50877 | 51820 | 49127 | 49636 | 51150 | 2.2% |
As a result, need testing solution is stable in 24 hours.
1.2.4.2 mobile phase ratio
Sample thief, adopts different mobile phase ratio to measure, the results are shown in Table 4.
The different mobile phase ratio test findings of table 4
Result shows, when mobile phase A changes in above-mentioned scope, in test sample chromatogram, parameters all meets the requirements, good tolerance.
1.2.4.3 chromatographic column
Sample thief, adopts the chromatographic column of different label to measure, the results are shown in Table 5.
The different chromatographic column test findings of table 5
| Tested number | Chromatographic column model | Measurement result (mg/g) |
| 1 | WondasilC18(5μm，4.6×250mm) | 0.0123 |
| 2 | DiamonsilC18(5μm，4.6×250mm) | 0.0130 |
Result shows, this law chromatographic column good tolerance.
diester-type alkaloids limit test in 1.3 3 batch samples
Get three batch samples
(140101 batches, 140102 batches, 140103 batches
), check the limit of diester-type alkaloids in accordance with the law, the results are shown in Table 6.
Table 6 three batch sample measurement result
| Lot number | 140101 | 140102 | 140103 |
| Result (μ g/ bag) | 23.9 | 24.5 | 25.6 |
Result shows, and in three batch samples, the check result of diester-type alkaloids is between 23.9 ~ 25.6 μ g/ bags.
[assay]
1 monkshood
With reference to " Chinese Pharmacopoeia " version (the second enlarged edition) mural nodules assay (monkshood, aconiti preparata,radix) and " Chinese Pharmacopoeia " version in 2010 high performance liquid chromatography (annex VI D) test in 2010.The content of monoester alkaloid (benzoylmesaconine, benzoyl aconine and benzoyl time aconine) in working sample.
1.1 assay method
Measure according to high performance liquid chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI D).
Chromatographic condition and system suitability: be connected the chromatographic column that phenyl bonded silica is filling agent with polarity ether; With the phosphoric acid solution of acetonitrile-0.1% (23 ﹕ 77) for mobile phase; Determined wavelength is 232nm.Number of theoretical plate calculates by benzoylmesaconine peak and is not less than 5000.
The preparation of reference substance solution gets benzoylmesaconine, benzoyl aconine, benzoyl time aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes the solution of every 1ml containing 0.2mg, product stock solution in contrast; Precision measures above-mentioned reference substance stock solution each 10ml, 5ml and 5ml respectively, puts in the measuring bottle of same 100ml, is diluted to scale, shakes up with the phosphoric acid solution of 0.1%, 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, scale is diluted to the phosphoric acid solution of 0.1%, shake up, be prepared into the reference substance solution of every 1ml containing benzoylmesaconine 15 μ g, benzoyl aconine 2 μ g, benzoyl time aconine 3 μ g.
Solid-phase extraction column system suitability: precision measures benzoylmesaconine, benzoyl aconine, the each 10ml of reference substance stock solution of benzoyl time 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, be placed in the solid-phase extraction column handled well (with the agent of mixed type cation exchange reverse phase absorption for filling agent, 150mg, 6ml, use acetonitrile successively in advance, the each 6ml wash-out of water) on, successively with water 3ml, the ammonia solution of 1.25%, water, methyl alcohol, the each 5ml wash-out of acetonitrile, after eluent flows to end, place 5 minutes, use the mixed solution 10ml wash-out of acetonitrile-strong ammonia solution (90 ﹕ 10) again, collect eluent, in less than 40 DEG C decompression and solvent recoveries to dry, residue adds mobile phase 5ml makes dissolving, filter, get subsequent filtrate as solid-phase extraction column system suitability solution.
Accurate absorption said system employment and suitability test (E & ST) solution and each 20 μ l of reference substance solution respectively, injection liquid chromatography, measure, computing system employment and suitability test (E & ST) solution and the ratio of corresponding Component peak area each in reference substance solution, be all not less than 0.95.
Attached sweet particle is got in the preparation of need testing solution, porphyrize, takes 0.5g, accurately weighed, put in tool plug conical flask, precision adds 0.1mol/L hydrochloric acid solution 25ml, close plug, weighed weight, ultrasonic process also jolting constantly in 40 minutes, let cool, more weighed weight, the weight of less loss is supplied with 0.1mol/L hydrochloric acid solution, shake up, leave 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 ", operating in accordance with the law, to obtain final product;
Determination method is accurate respectively draws reference substance solution and each 20 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
1.2 CP method and method monoester alkaloid content of the present invention measure applicability and compare
Monkshood monoester alkaloid content determination method (hereinafter referred to as " CP method ") and monkshood monoester alkaloid content determination method of the present invention under " Chinese Pharmacopoeia " version in 2010 second enlarged edition fugui gutong keli item, the applicability measured for attached sweet granule monkshood monoester alkaloid content compares.
1.2.1 " CP method " measures attached sweet granule monoester alkaloid employment and suitability test (E & ST)
Chromatographic condition and system suitability are that the chromatographic column of filling agent is (hereinafter referred to as C with octadecylsilane chemically bonded silica
18post); With acetonitrile-0.1% phosphoric acid solution (22:78) for mobile phase; Determined wavelength is 232nm.Number of theoretical plate calculates by benzoylmesaconine peak and is all not less than 10000.
The preparation of reference substance solution: get benzoylmesaconine, benzoyl aconine, benzoyl time aconine reference substance in right amount, accurately weighed, add acetonitrile respectively and make the solution of every 1ml containing 0.2mg, product stock solution in contrast; Precision measures each reference substance stock solution 15ml, 2ml, 3ml respectively, put in the measuring bottle of same 200ml, add acetonitrile to 40ml, scale is diluted to the phosphoric acid solution of 0.1%, shake up, be prepared into the reference substance solution of every 1ml containing benzoylmesaconine 15 μ g, benzoyl aconine 2 μ g, benzoyl time aconine 3 μ g.
The preparation of need testing solution: get attached sweet particle porphyrize, get 0.5g, accurately weighed, put in tool plug conical flask, precision adds 0.1mol/L hydrochloric acid solution 25ml, close plug, weighed weight, ultrasonic process also jolting constantly in 40 minutes, 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, be added in solid-phase extraction column (with the agent of mixed type cation exchange reverse phase absorption for filling agent, 150mg, capacity is 6ml, use acetonitrile successively in advance, the each 6ml wash-out of water) on, successively with water 3ml, ammonia solution (5 → 100), water, methyl alcohol, the each 5ml wash-out of acetonitrile, after eluent flows to end, place 5 minutes, continue with the mixed solution 10ml wash-out of acetonitrile-strong ammonia solution (90:10), collect eluent, in less than 40 DEG C decompression and solvent recoveries to dry, the mixed solution 5ml that residue precision adds acetonitrile-0.1% phosphoric acid solution (20:80) makes dissolving, filter, get subsequent filtrate, obtain.
Determination method: accurate absorption reference substance solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.Determine the C of 3 different manufacturers
18post, need testing solution tailing factor the results are shown in Table 7.
The different label C of table 7
18post tailing factor measurement result
Result shows, when " CP method " measures this product monoester type alkaloid, and the C of 3 different labels
18post, benzoylmesaconine, benzoyl time aconine, benzoyl time aconine chromatographic peak all trail seriously, and peak type is poor, affects the Measurement accuracy of peak area, therefore, C
18post 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, determination method are all constant, just chromatographic column is changed into and connect chromatographic column that phenyl bonded silica is filling agent (hereinafter referred to as phenyl post) with polarity ether, assay is carried out to attached sweet particle monoester alkaloid.Determine the phenyl post of 3 different manufacturers, need testing solution tailing factor the results are shown in Table 8:
Table 8 different label phenyl post tailing factor measurement result
Result shows, benzoylmesaconine, benzoyl time aconine, benzoyl time aconine chromatographic peak tailing factor are all 0.95 ~ 1.05, chromatographic peak peak shape is good, and system suitability is good, shows 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 attached sweet granule monoester alkaloid specificity test
Get attached sweet granule and not containing the negative granules agent of monkshood, test according to " CP method " described liquid-phase condition and need testing solution preparation method.As a result, need testing solution chromatogram is having chromatographic peak with reference substance solution chromatogram corresponding position, but peak shape is poor, and hangover is serious, and benzoylmesaconine chromatographic peak degree of separation is less than 1.5; Negative sample solution is having chromatographic peak to detect with benzoylmesaconine reference substance chromatogram corresponding position, shows that " CP method " measures monoester alkaloid specificity in attached sweet particle poor.Detailed results is in table 9.
Table 9 " CP method " specificity result
1.3.2 the inventive method measures attached sweet granule monoester alkaloid specificity test
Separately get attached sweet granule and not containing the negative granules agent of monkshood, test according to by liquid-phase condition described in the inventive method and need testing solution preparation method.Result, need testing solution chromatogram is having chromatographic peak with reference substance solution chromatogram corresponding position, and peak shape is good, and degree of separation is good, negative sample solution is detecting without chromatographic peak with reference substance solution corresponding position, shows that the inventive method measures monoester alkaloid specificity in attached sweet particle good.Detailed results is in table 10.
Table 10 the inventive method specificity result
1.4 monoester alkaloid content mensuration methodology researchs of the present invention
1.4.1 solid-phase extraction column system suitability
Get benzoylmesaconine, benzoyl aconine, benzoyl time aconine reference substance, carry out solid-phase extraction column system suitability according to said method.
Result: solid-phase extraction column system suitability solution is all greater than 0.95 to corresponding Component peak area ratio each in reference substance solution, and detailed results is in table 11.
Table 11 solid-phase extraction column system suitability result
| Benzoylmesaconine | Benzoyl aconine | Benzoyl time aconine | |
| Reference substance solution | 546918 | 264040 | 307307 |
| Need testing solution | 541539 | 264615 | 304233 |
| Ratio | 0.99 | 1.00 | 0.99 |
1.4.2 accuracy experiment
The preparation of reference substance stock solution: get benzoylmesaconine reference substance appropriate, accurately weighed, make the solution of every 1ml containing 3.7 μ g with 0.1mol/L hydrochloric acid solution, to obtain final product.
The preparation of need testing solution: the attached sweet particle 0.25g getting porphyrize, 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 determination method, with described in the inventive method, measure, and calculate the recovery, the results are shown in Table 12.
Table 12 accuracy test result
As a result, accuracy is good.
1.4.3 precision
1.4.3.1 repeated
Get the attached sweet particle 0.5g of porphyrize, accurately weighed 6 parts, measure according to the method for the invention, the results are shown in Table 13.
Table 13 replica test result
| Tested number | 1 | 2 | 3 | 4 | 5 | 6 | RSD(%) |
| Result (mg/g) | 0.4424 | 0.4426 | 0.4395 | 0.4415 | 0.4410 | 0.4392 | 0.32 |
Result shows, repeatability is good.
1.4.3.2 different instrument
On different instrument, same test sample is measured respectively, investigate the impact of instrument variation on precision, the results are shown in Table 14.
The different instrument test result of table 14
As a result, between different instrument, precision is good.
1.4.3.3 different tests personnel
By different personnel, same test sample is measured, investigate the impact of personnel's variation on precision, the results are shown in Table 15.
The different personnel's test findings 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 is measured respectively by same analyst, investigate the impact of date variation on precision, the results are shown in Table 16.
Table 16 different time test findings
As a result, between same date, precision is not good.
1.4.4 durability
1.4.4.1 stability of solution
Get need testing solution room temperature to place, draw 20 μ l, injection liquid chromatography respectively at the 0th, 3,6,9,12 hour, measure, record chromatographic peak area, the results are shown in Table 17.
Table 17 solution stability testing
| Standing time (hour) | 0 | 3 | 6 | 9 | 12 | RSD |
| Benzoylmesaconine | 376669 | 375633 | 375647 | 374682 | 375416 | 0.19% |
| Benzoyl aconine | 36058 | 36014 | 35985 | 36207 | 36009 | 0.25% |
| Benzoyl time aconine | 71361 | 71082 | 70975 | 70650 | 69394 | 1.1% |
As a result, need testing solution is stable in 12 hours.
1.4.4.2 determined wavelength
Get this product need testing solution, measure under 227nm, 232nm, 237nm wavelength respectively, the results are shown in Table 18.
Table 18 different wave length test findings
As a result, wavelength good tolerance.
1.4.4.3 flow velocity
Get this product need testing solution, adopt 0.8ml/min, 1.0ml/min, 1.2ml/min flow velocity to measure respectively, the results are shown in Table 19.
Table 19 different in flow rate test findings
as a result, flow velocity good tolerance.
1.4.4.4 mobile phase ratio
Get this product need testing solution, adopt different mobile phase ratio to measure, the results are shown in Table 20.
The different mobile phase ratio test findings of table 20
As a result, mobile phase ratio good tolerance.
1.4.4.5 column temperature
Get this product need testing solution, adopt 25 DEG C, 30 DEG C, 35 DEG C column temperatures to measure respectively, the results are shown in Table 21.
The different column temperature test findings of table 21
As a result, column temperature good tolerance.
1.4.4.6 chromatographic column label
Get this product need testing solution, adopt the chromatographic column of different label to measure, the results are shown in Table 22.
The different chromatographic column test findings of table 22
As a result, chromatographic column good tolerance.
1.4.5, linearly
1.4.5.1 benzoylmesaconine
Get benzoylmesaconine reference substance appropriate, accurately weighed, prepare the solution of every 1ml 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).The each 20 μ l of the above-mentioned reference substance solution of accurate absorption respectively, injection liquid chromatography, record chromatographic peak area.With benzoylmesaconine reference substance solution concentration for horizontal ordinate, with corresponding peak area for ordinate, 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
Get benzoyl aconine reference substance appropriate, accurately weighed, prepare the solution of every 1ml 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).The each 20 μ l of the above-mentioned reference substance solution of accurate absorption respectively, injection liquid chromatography, record chromatographic peak area.With benzoyl aconine reference substance solution concentration for horizontal ordinate, with corresponding peak area for ordinate, 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 time aconine
Get benzoyl time aconine reference substance appropriate, accurately weighed, prepare the solution of every 1ml 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).The each 20 μ l of the above-mentioned reference substance solution of accurate absorption respectively, injection liquid chromatography, record chromatographic peak area.With benzoyl time aconine reference substance solution concentration for horizontal ordinate, with corresponding peak area for ordinate, carry out linear regression, obtain equation of linear regression: y=23916x-3973.3, r=0.9995.
As a result, benzoyl time aconine is good linear relationship with peak area between 0.8 ~ 8.0 μ g/ml.
1.4.6 scope
Take this product content by 80%, 100%, 120% of 0.5g respectively, prepare need testing solution, measure monoester alkaloid content, the results are shown in Table 23.
Table 23 scope test findings
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
Get three batches quick particles
(140101 batches, 140102 batches, 140103 batches
), measure monoester alkaloid content, the results are shown in Table 24.
Table 24 three batch sample assay result
| Lot number | 140101 batches | 140102 batches | 140103 batches |
| Result (μ g/ bag) | 778 | 801 | 851 |
2 Radix Glycyrrhizaes
With reference to " method under Chinese Pharmacopoeia 2005 version () and " Chinese Pharmacopoeia " version (one) licorice medicinal materials assay item in 2010 is tested, and measures the content of liquiritin in developing products.
2.1 method
Chromatographic condition and system suitability: the chromatographic column taking octadecylsilane chemically bonded silica as filling agent; With acetonitrile-0.5% glacial acetic acid (18 ﹕ 82) for mobile phase; Determined wavelength is 276nm; Number of theoretical plate calculates should be not less than 4000 by liquiritin peak.
Reference substance solution to prepare extracting Radix Glycyrrhizae glycosides reference substance appropriate, accurately weighed, add 70% ethanol and make the solution of every 1ml containing 20 μ g, to obtain final product.
Attached sweet particle porphyrize is got in the preparation of need testing solution, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic process 30 minutes, lets cool, weighed weight again, supply the weight of less loss with 70% ethanol, shake up, filter, get subsequent filtrate, to obtain final product.
Determination method is accurate respectively draws above-mentioned reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
2.2 Method validation
2.2.1 specificity
The preparation of need testing solution is got 130901 batches quick particles and not containing the negative preparation of Radix Glycyrrhizae, is prepared need testing solution according to said method.
Measure according to above-mentioned liquid-phase condition and determination method, result, in test sample chromatogram with chromatographic peak reference substance chromatogram relevant position having identical retention time, degree of separation is good, negative sample solution detects without chromatographic peak in relevant position, show negative noiseless, it is good that this law measures Radix Glycyrrhizae specificity.
2.2.2 accuracy
Accuracy test contrast solution to prepare extracting Radix Glycyrrhizae glycosides reference substance appropriate, accurately weighed, add 70% ethanol and make the reference substance solution of every 1ml containing 0.1mg.
130901 batches of content porphyrizes are got in the preparation of accuracy test need testing solution, get 0.25g, accurately weighed, totally 6 parts, precision adds above-mentioned liquiritin 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 repeated
Get 130901 batches of content porphyrizes, get 0.5g, accurately weighed, totally 6 parts, by preferred need testing solution preparation method operation, measure liquiritin content respectively.The results are shown in Table 26.
Table 26 replica test result
| Tested number | 1 | 2 | 3 | 4 | 5 | 6 | RSD |
| Result (mg/g) | 1.68 | 1.64 | 1.70 | 1.68 | 1.64 | 1.66 | 1.49% |
As a result, repeatability is good.
2.2.3.2 different personnel
Get 130901 batches quick particles, measure liquiritin content by different personnel respectively, investigate the impact of personnel's variation on precision, the results are shown in Table 27.
The different personnel's test findings of table 27
As a result, the precision of different personnel's test is good.
2.2.3.4 different instrument
Get 130901 batches quick particles, on different instrument, measure liquiritin content respectively, investigate the impact of instrument variation on precision, the results are shown in Table 28.
The different instrument test result of table 28
As a result, the precision of different instrument test is good.
2.2.3.5 different time
Get 130901 batches of quick particles, measure liquiritin content at not same date respectively, investigate the impact of date variation on precision.The results are shown in Table 29.
Table 29 different time test findings
As a result, the precision of different time test is good.
2.2.4 linear
Extracting Radix Glycyrrhizae glycosides reference substance is appropriate, and making concentration respectively with 70% ethanol is the reference substance solution of every 1ml containing liquiritin 8,16,32,48,64 μ g; The each 10 μ l of the above-mentioned reference substance solution of accurate absorption respectively, injection liquid chromatography, record chromatogram.With liquiritin reference substance solution concentration for horizontal ordinate, take peak area as ordinate, carry out linear regression, obtaining regression equation is: y=19203x+2039.4, r=0.9997.
Result shows, liquiritin is good linear relationship with peak area between 8 ~ 64 μ g/ml.
2.2.5 durability
2.2.5.1 stability of solution
Get 130901 batches quick particles and prepare need testing solution, room temperature is placed, and respectively at the 0th, 5,10,15,20 hour, accurate absorption 10 μ l, injection liquid chromatography, record liquiritin chromatographic peak area, the results are shown in Table 30.
Table 30 solution stability testing result
| Standing time (hour) | 0 | 15 | 20 | RSD |
| Peak area | 516740 | 509471 | 505117 | 1.15% |
As a result, need testing solution is stable in 20 hours.
2.2.5.2 column temperature
Get 130901 batches quick particles and prepare need testing solution, adopt 25 DEG C, 30 DEG C, 35 DEG C column temperature analyses respectively, measure liquiritin content, the results are shown in Table 31.
The different column temperature test findings of table 31
As a result, the good tolerance of different column temperature test.
2.2.5.3 flow velocity
Get 130901 batches quick capsules and prepare need testing solution, adopt the analysis of 0.8ml/min, 1.0ml/min, 1.2ml/min flow velocity respectively, measure liquiritin content.
Table 32 different in flow rate test findings
As a result, the good tolerance of mobile phase different in flow rate test.
2.2.5.4 determined wavelength
Get 130901 batches quick particles and prepare need testing solution, analyze under 227nm, 232nm, 237nm wavelength respectively, measure liquiritin content, the results are shown in Table 33.
Table 33 different wave length test findings
As a result, the good tolerance of different determined wavelength test.
2.2.5.5 mobile phase ratio
Get 130901 batches quick particles and prepare need testing solution, adopt different mobile phase ratio analysis respectively, measure liquiritin content, the results are shown in Table 34.
The different mobile phase ratio test findings of table 34
As a result, the good tolerance of different mobile phase ratio test.
2.2.5.6 chromatographic column
Get 130901 batches quick particles and prepare need testing solution, adopt the chromatogram column analysis of different label, measure liquiritin content, the results are shown in Table 35.
The different chromatographic column test findings of table 35
As a result, the good tolerance of different chromatographic column test.
2.2.6 scope
Get 130901 batches quick granular contents porphyrizes, press 50% of sampling amount 0.5g, 100%, 150% accurate title respectively and measure liquiritin content, the results are shown in Table 36.
Table 36 scope test findings
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 liquiritin in 2.3 3 batch samples
Get three batch samples (140101 batches, 140102 batches, 140103 batches), measure liquiritin content in accordance with the law, the results are shown in Table 37.
The result of liquiritin assay in table 37 three batch sample
| Lot number | 140101 | 140102 | 140103 |
| Result (mg/ grain) | 3.22 | 3.48 | 3.63 |
As a result, in three batch samples the content of liquiritin between 3.22 ~ 3.63mg/ grain.
Embodiment
embodiment 1: attached sweet granule monoester alkaloid content measures
1) granule is prepared: get monkshood 6kg, Radix Glycyrrhizae 1kg, boiling twice, first time adds 8 times of water gagings, decocts 2 hours, and second time adds 6 times of water gagings, decoct 1 hour, merge twice decocting liquid, filter, concentrated, drying under reduced pressure, pulverizes, and it is appropriate to add dextrin, mixing, wet granulation, dry, whole grain, obtains the granule of attached sweet medicine.
2) check
Sample thief, test according to quality standard of the present invention, assay sees the following form:
table 38 assay
Embodiment 2: attached sweet granule monoester alkaloid content measures
1) granule is prepared: get monkshood 18kg, Radix Glycyrrhizae 9kg, boiling twice, first time adds 10 times of water gagings, decocts 1.5 hours, and second time adds 8 times of water gagings, decoct 1 hour, merge twice decocting liquid, filter, concentrated, drying under reduced pressure, pulverizes, and it is appropriate to add dextrin, mixing, wet granulation, dry, whole grain, obtains the granule of attached sweet medicine.
2) check
Sample thief, test according to quality standard of the present invention, assay sees the following form:
table 39 assay
Embodiment 3: attached sweet granule monoester alkaloid content measures
1) prepare granule: get monkshood 10kg, Radix Glycyrrhizae 5kg, boiling three times, first time adds 8 times of water gagings, decoct 1.5 hours, second time adds 8 times of water gagings, decocts 1 hour, third time adds 6 times of water gagings, decocts 1 hour, merges three decocting liquids, filter, concentrated, drying under reduced pressure, pulverize, add dextrin appropriate, mixing, wet granulation, drying, whole grain, obtains the granule of attached sweet medicine.
2) check
Sample thief, test according to quality standard of the present invention, assay sees the following form:
table 40 assay
Embodiment 4: attached sweet tablet monoester alkaloid content measures
1) tablet is prepared: get monkshood 10kg, Radix Glycyrrhizae 4kg, boiling 3 times.Add the water of 10 times amount of crude drug quality for the first time, decoct 2h, second and third time respectively adds the water of 8 times amount respectively, decoct 1.5h respectively, merge 3 decoction liquor, filter, getting filtrate reduced in volume to relative density is 1.12(60 DEG C), add ethanol, just add and just stir and evenly mix, alcohol content is made to reach 70%, leave standstill 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 it is appropriate to add starch, granulate, add talcum powder appropriate, mixing, compressing tablet, sugar coating, obtains the tablet of attached sweet medicine.
2) check
Sample thief, test according to quality standard of the present invention, assay sees the following form:
table 41 assay
Embodiment 5: attached sweet capsule monoester alkaloid content measures
1) capsule is prepared: get monkshood 18kg, Radix Glycyrrhizae 6kg, boiling twice, first time adds 8 times of water gagings, decoct 2 hours, second time adds 6 times of water gagings, decoct 1 hour, merge twice decocting liquid, filter, getting filtrate reduced in volume to relative density is 1.15(60 DEG C), add ethanol, just add and just stir and evenly mix, alcohol content is made to reach 80%, leave standstill 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 superfine silica gel powder appropriate, granulate, drying under reduced pressure, incapsulate, obtain the capsule of attached sweet medicine.
2) check:
Sample thief, test according to quality standard of the present invention, assay sees the following form:
table 42 assay
Claims (2)
1. an attached sweet drug quality detection method, it is characterized in that: adopt high performance liquid chromatography to differentiate monkshood, adopt thin-layered chromatography to differentiate Radix Glycyrrhizae, adopt high performance liquid chromatography to control diester-type alkaloids, adopt high effective liquid chromatography for measuring monkshood and Radix Glycyrrhizae content.
2. the attached sweet drug quality detection method of one according to claim 1, is characterized in that:
[proterties]this product content be brown color to sepia, micro-sweet, the micro-hardship of taste;
[discriminating](1) monkshood is differentiated: according to the method test under monkshood assay item, the chromatographic peak corresponding with benzoylmesaconine, benzoyl aconine and benzoyl time aconine reference substance chromatographic peak retention time should be presented in test sample chromatogram, namely determine it is the preparation containing monkshood;
(2) Radix Glycyrrhizae is differentiated: get this product content 0.5 ~ 2.5g, add diethyl ether 40ml, adds hot reflux 1 hour, filter, discard ether liquid, the dregs of a decoction add methyl alcohol 30ml, add hot reflux 1 hour, filter, filtrate evaporate to dryness, the residue 40ml that adds water makes dissolving, extracts 3 times, each 20ml with normal butyl alcohol, merge normal butyl alcohol liquid, wash 3 times with water, discard water liquid, normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Another extracting Radix Glycyrrhizae control medicinal material 1g, is made in the same way of control medicinal material solution; Extracting Radix Glycyrrhizae glycosides reference substance again, adds methyl alcohol and makes the solution of every 1ml containing 2mg, product solution in contrast; According to the annex VI B thin-layered chromatography test of " Chinese Pharmacopoeia " version in 2010, draw each 5 μ l of above-mentioned three kinds of solution, put on the silica gel g thin-layer plate prepared in same use 1% sodium hydroxide solution respectively, with Zheng Ding Chun ﹕ Nong An Shi Ye ﹕ ethanol=5 ﹕ 2 ﹕ 1 for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear, inspects under putting 365nm ultraviolet lamp; In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material, the fluorescence spot of aobvious same color, namely determines it is the preparation containing Radix Glycyrrhizae;
[inspection]diester-type alkaloids: according to " Chinese Pharmacopoeia " version in 2010 annex VI D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With Yi Jing ﹕ tetrahydrofuran=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 determined wavelength is 232nm; Number of theoretical plate calculates should be not less than 30000 by aconitine peak;
Table 1 gradient elution program
Mesaconine reference substance is got in the preparation of reference substance solution, Hypaconitine reference substance, aconitine reference substance are appropriate, accurately weighed, add acetonitrile respectively and make the solution of every 1ml containing 0.2mg, product stock solution in contrast, 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, and precision measures 5ml, is placed on the solid-phase extraction column handled well, solid-phase extraction column with the agent of mixed type cation exchange reverse phase absorption for filling agent, 150mg, capacity is 6ml, uses each 6ml wash-out of acetonitrile, water in advance successively; Successively with 0.lmol/L hydrochloric acid solution, methyl alcohol, each 5ml wash-out of acetonitrile, discard eluent, place 5 minutes, continue with the mixed solution 10ml wash-out of Yi Jing ﹕ strong ammonia solution=90 ﹕ 10, collect eluent, in less than 40 DEG C decompression and solvent recoveries to dry, the mixed solution 3ml that residue precision adds Yi Jing ﹕ 0.1% phosphoric acid solution=30 ﹕ 70 makes dissolving, filter, get subsequent filtrate as solid-phase extraction column system suitability solution;
Accurate absorption above-mentioned solid phase extraction column system suitability solution and each 10 μ l of reference substance solution respectively, injection liquid chromatography, measure, computing system employment and suitability test (E & ST) solution and each corresponding Component peak area ratio of reference substance solution, must not be less than 0.95;
The preparation of need testing solution: it is appropriate to get this product, porphyrize, get 1 ~ 3g, accurately weighed, put in tool plug conical flask, precision adds the mixed solution 50ml of Yi Jing ﹕ strong ammonia solution=90 ﹕ 10, close plug, weighed weight, ultrasonic process 30 minutes, the weight of less loss is supplied with the mixed solution of Yi Jing ﹕ 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 " be placed in handle well solid-phase extraction column on ", operate in accordance with the law, obtain,
Determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product;
otherevery regulation relevant under should meeting " Chinese Pharmacopoeia " version in 2010 annex rules of preparations item;
[assay]monkshood assay: according to " Chinese Pharmacopoeia " version in 2010 annex VI D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: be connected the chromatographic column that phenyl bonded silica is filling agent with polarity ether; With phosphoric acid solution=23 ﹕ 77 of Yi Jing ﹕ 0.1% for mobile phase; Determined wavelength is 232nm, and number of theoretical plate calculates by benzoylmesaconine peak and is not less than 5000;
The preparation of reference substance solution gets benzoylmesaconine, benzoyl aconine, benzoyl time aconine reference substance in right amount, accurately weighed, adds acetonitrile respectively and makes the solution of every 1ml containing 0.2mg, product stock solution in contrast; Precision measures above-mentioned reference substance stock solution each 10ml, 5ml and 5ml respectively, put in the measuring bottle of same 100ml, scale is diluted to 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, scale is diluted to the phosphoric acid solution of 0.1%, shake up, be prepared into the reference substance solution of every 1ml containing benzoylmesaconine 15 μ g, benzoyl aconine 2 μ g, benzoyl time aconine 3 μ g;
Solid-phase extraction column system suitability precision measures reference substance stock solution each 10ml, 5ml and 5ml of benzoylmesaconine, benzoyl aconine, benzoyl time aconine, 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, be placed on the solid-phase extraction column handled well, this solid-phase extraction column with the agent of mixed type cation exchange reverse phase absorption for filling agent, specification is 150mg, 6ml, uses each 6ml wash-out of acetonitrile, water in advance successively; Successively with water 3ml, ammonia solution, water, methyl alcohol, each 5ml wash-out of acetonitrile of 1.25%, after eluent flows to end, place 5 minutes, then use the mixed solution 10ml wash-out of Yi Jing ﹕ 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, gets subsequent filtrate as solid-phase extraction column system suitability solution;
Accurate absorption said system employment and suitability test (E & ST) solution and each 20 μ l of reference substance solution respectively, injection liquid chromatography, measure, computing system employment and suitability test (E & ST) solution and the ratio of corresponding Component peak area each in reference substance solution, be all not less than 0.95;
Attached sweet particle is got in the preparation of need testing solution, porphyrize, takes 0.2 ~ 0.6g, accurately weighed, put in tool plug conical flask, precision adds 0.1mol/L hydrochloric acid solution 25ml, close plug, weighed weight, ultrasonic process also jolting constantly in 40 minutes, let cool, more weighed weight, the weight of less loss is supplied with 0.1mol/L hydrochloric acid solution, shake up, leave 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 ", operating in accordance with the law, to obtain final product;
Determination method is accurate respectively draws reference substance solution and each 20 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;
Radix Glycyrrhizae assay: according to " Chinese Pharmacopoeia " version in 2010 annex VI D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: the chromatographic column taking octadecylsilane chemically bonded silica as filling agent; With Yi Jing ﹕ 0.5% glacial acetic acid solution=18 ﹕ 82 for mobile phase; Determined wavelength is 276nm; Number of theoretical plate calculates should be not less than 4000 by liquiritin peak;
The preparation of reference substance solution: extracting Radix Glycyrrhizae glycosides reference substance is appropriate, accurately weighed, adds 70% ethanol and makes the solution of every 1ml containing 20 μ g, to obtain final product;
The preparation of need testing solution: get this product content, porphyrize, get 0.2 ~ 0.6g, accurately weighed, put in tool plug conical flask, precision adds 70% ethanol 50ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 70% ethanol, shake up, filter, get subsequent filtrate, to obtain final product;
Measure: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
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| CN113092655A (en) * | 2021-04-12 | 2021-07-09 | 长春中医药大学 | Method for detecting effective components in aconite decoction by high performance liquid chromatography |
| CN114910576A (en) * | 2022-04-07 | 2022-08-16 | 国药集团广东环球制药有限公司 | Method for detecting aconite monoester type alkaloid component in cassia twig, peony and rhizoma anemarrhenae decoction |
| WO2024007538A1 (en) * | 2022-07-06 | 2024-01-11 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Content measurement method for six alkaloid components in small meridian-activating pill |
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| CN113092655A (en) * | 2021-04-12 | 2021-07-09 | 长春中医药大学 | Method for detecting effective components in aconite decoction by high performance liquid chromatography |
| CN114910576A (en) * | 2022-04-07 | 2022-08-16 | 国药集团广东环球制药有限公司 | Method for detecting aconite monoester type alkaloid component in cassia twig, peony and rhizoma anemarrhenae decoction |
| WO2024007538A1 (en) * | 2022-07-06 | 2024-01-11 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Content measurement method for six alkaloid components in small meridian-activating pill |
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