CN103316102B - Detection method for external traditional Chinese medicine preparation for treating haemorrhoids - Google Patents
Detection method for external traditional Chinese medicine preparation for treating haemorrhoids Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a detection method for an external traditional Chinese medicine preparation for treating haemorrhoids. The external traditional Chinese medicine preparation for treating haemorrhoids is prepared from the following raw materials in parts by weight: 105-135 parts of fineleaf schizonepeta herb, 105-135 parts of radix saposhnikoviae, 270-330 parts of garden balsam stem, 80-100 parts of radix aconiti, 270-330 parts of bothriospermum chinense, 80-100 parts of radix aconiti kusnezoffii and 105-135 parts of radix sophorae flavescentis. The detection method for the external traditional Chinese medicine preparation for treating haemorrhoids comprises identification of contents and measurement of component content, wherein the identification of contents is the identification of fineleaf schizonepeta herb, radix saposhnikoviae, monkshood and radix aconiti kusnezoffii by thin layer chromatography; and the measurement of component content is the measurement of the content of sophocarpidine and aconitum alkaloids by high performance liquid chromatography. The method provided by the invention is simple, convenient, quick and accurate, has good sensitivity and high stability, improves the effective control on the drug quality, and guarantees the quality.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to a kind of detection method for the treatment of hemorrhoid Chinese medicine for outer use.
Background technology
Compound schizonepeta fumigation and wash method preparation, as a kind for the treatment of hemorrhoid Chinese medicine for outer use having gone on the market, has dispeiling pathogenic wind and removing dampness, the effect of reducing swelling and alleviating pain.Be mainly used in clinically the diseases such as external hemorrhoid, mixed hemorrhoid, interior prolapse of hemorrhoid incarceration, anal fissure, perianal abscess or anal fistula acute attack; But existing compound schizonepeta fumigation and wash method standard preparation falls behind, detection index is less, only there is the simple discriminating of general medical material, because containing toxic medical material Radix Aconiti Kusnezoffii, Radix Aconiti in prescription, there is no the index restriction of assay yet, the quality determining method of prior art can not effectively be controlled the quality of compound schizonepeta fumigation and wash method preparation, thereby will affect production testing and the drug safety of product.
Summary of the invention
The invention provides a kind of detection method for the treatment of hemorrhoid Chinese medicine for outer use, can control well the stable homogeneous of product quality, ensure product quality.
Treatment hemorrhoid Chinese medicine for outer use of the present invention is to be made up of the raw material of following parts by weight:
270～330 parts of 105～135 parts of Herba speranskiae tuberculataes of 105～135 parts of Radix Saposhnikoviaes of Herba Schizonepetae
80～100 parts of 270～330 portions of Radix Aconiti Kusnezoffii of 80～100 portions of Herba Potentillae Chinensis of Radix Aconiti
105～135 parts of Radix Sophorae Flavescentiss.
Optimum feed stock consists of:
300 parts of 120 parts of Herba speranskiae tuberculataes of 120 parts of Radix Saposhnikoviaes of Herba Schizonepetae
90 parts of 300 portions of Radix Aconiti Kusnezoffii of 90 portions of Herba Potentillae Chinensis of Radix Aconiti
120 parts of Radix Sophorae Flavescentiss.
Concrete preparation process is as follows:
First Herba Schizonepetae, Radix Aconiti, Radix Aconiti Kusnezoffii are ground into fine powder, then all the other raw materials are decocted with water 3 times, decocting time is respectively 2,2,1 hours, collecting decoction, filter, filtrate decompression is concentrated, dry, pulverize into fine powder, finally two kinds of fine powders are merged and mixed, granulate, be drying to obtain.
The detection method for the treatment of hemorrhoid Chinese medicine for outer use of the present invention, comprise the discriminating of content and the assay that contains composition, the discriminating of content is that thin layer chromatography is differentiated Herba Schizonepetae, Radix Saposhnikoviae, Radix Aconiti, Radix Aconiti Kusnezoffii composition, and the assay that contains composition is the content of high effective liquid chromatography for measuring matrine, aconite alkaloids.
The discrimination method concrete steps of described Herba Schizonepetae are as follows:
Sample thief porphyrize, takes 6g, adds acetone 30ml supersound process 30 minutes, filter, and filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, as need testing solution; Get Herba Schizonepetae reference substance 1.0g, add acetone 20ml supersound process 30 minutes, filter, filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, product solution in contrast;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution and the each 5 μ l of reference substance solution, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, cyclohexane extraction-ethyl acetate taking volume ratio as 7:3 is developing solvent, launch, take out, dry, put under 365nm ultra-violet lamp and inspect; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, aobvious identical red fluorescence speckle.
The discrimination method concrete steps of described Radix Saposhnikoviae are as follows:
Sample thief porphyrize, takes 6g, adds acetone 30ml supersound process 30 minutes, filter, and filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, as need testing solution; Get Radix Saposhnikoviae reference substance 1g, add acetone 20ml, supersound process 30 minutes, filters, filtrate evaporate to dryness, and residue adds ethanol 1ml to be made to dissolve, product solution in contrast;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution 10 μ l and reference substance solution 5 μ l, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, ethyl acetate-ethanol taking volume ratio as 4:1 is developing solvent, launch, take out, dry, put under 254nm ultra-violet lamp and inspect; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the fluorescence speckle of aobvious same color.
Described Radix Aconiti, the discrimination method concrete steps of Radix Aconiti Kusnezoffii are as follows:
Sample thief porphyrize, takes 5g, adds dehydrated alcohol 30ml supersound process 30 minutes, filter, dehydrated alcohol 5ml washing 2 times for filtering residue, merges with filtrate, evaporate to dryness, residue all dissolves with 10% hydrochloric acid solution 20ml, filters, filtrate is adjusted pH value 9～10 with ammonia, with ether jolting extraction 2 times, each 20ml, extracting solution low temperature evaporate to dryness, residue adds dehydrated alcohol 1ml to be made to dissolve, as need testing solution; Get aconitine reference substance, make every 1ml containing the solution of 1mg, product solution in contrast with dehydrated alcohol;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution and the each 10 μ l of reference substance solution, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, ether-ethyl acetate-ammonia taking volume ratio as 5:5:1 is developing solvent, launch, take out, dry, spray is with rare bismuth potassium iodide solution; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
The content assaying method concrete steps of described matrine are as follows:
Reference " Chinese Pharmacopoeia " 2010 annex VI D high effective liquid chromatography for measuring:
(1) chromatographic condition and system suitability: with octadecylsilane chemically bonded silica be filler; Volume ratio is acetonitrile-0.05mol/L KH of 5:95:0.8:0.8
2pO
4-phosphoric acid-triethylamine is mobile phase; Detect wavelength 205nm;
(2) preparation of reference substance solution: get matrine reference substance, accurately weighed, add mobile phase and make the solution of every 1ml containing matrine 130 μ g, to obtain final product;
(3) preparation of need testing solution: sample thief porphyrize, get 0.25g, accurately weighed, put in tool plug conical flask, add strong ammonia solution 1ml and ether 30ml, close plug, hold over night, power is 250W, the frequency supersound process that is 33kHz 30 minutes, let cool, filter, container and residue divide 2 washings with ether 15ml, washing liquid and filtrate merge, evaporate to dryness, and residue adds the mutual-assistance of flowing and dissolves, be transferred in 25ml measuring bottle, be diluted to scale, shake up, filter, get subsequent filtrate, to obtain final product;
(4) algoscopy: accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.
The content assaying method concrete steps of described aconite alkaloids are as follows:
Reference " Chinese Pharmacopoeia " 2010 annex VI D high effective liquid chromatography for measuring:
(1) chromatographic condition and system suitability: with octadecylsilane chemically bonded silica be filler; Volume ratio is that acetonitrile-oxolane-0.1mol/L Spirit of Mindererus. (every 1000ml adds glacial acetic acid 0.5ml) of 55:33:312 is mobile phase; Detection wavelength is 230nm; Number of theoretical plate calculates and should be not less than 4000 by aconitine peak;
(2) preparation of reference substance solution: get aconitine, hypaconitine, mesaconitine reference substance, accurately weighed, the mixed solution that adds volume ratio and be hydrochloric acid-methanol of 1:100 is made the mixed solution of every 1ml containing aconitine, hypaconitine, the each 30 μ g of mesaconitine, to obtain final product;
(3) preparation of need testing solution: sample thief porphyrize, get 1g, accurately weighed, put in tool plug conical flask, it is the mixed solution 50ml of hydrochloric acid-methanol of 1:100 that precision adds volume ratio, weighed weight, water temperature is controlled at 20 DEG C, and power is 250W, the frequency supersound process that is 50kHz 40 minutes, lets cool, weighed weight again, the mixed solution of the hydrochloric acid-methanol that is 1:100 by volume ratio is supplied the weight of less loss, shakes up, and filters, get subsequent filtrate, to obtain final product;
(4) algoscopy: accurate reference substance solution and the each 20ul of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.
The present invention compared with prior art, has following beneficial effect:
Detection method of the present invention is easy, quick, accurate, and sensitivity is good, stability is high, has improved the effective control to drug quality, has ensured quality.
Brief description of the drawings
Fig. 1 is matrine standard curve.
Fig. 2 is the high-efficient liquid phase chromatogram of matrine reference substance.
Fig. 3 is the high-efficient liquid phase chromatogram of Radix Sophorae Flavescentis negative sample.
Fig. 4 is the high-efficient liquid phase chromatogram of Sophora flavescens.
Fig. 5 is the high-efficient liquid phase chromatogram of compound schizonepeta fumigation and washing agent sample.
Fig. 6 is the high-efficient liquid phase chromatogram of aconitine, hypaconitine, mesaconitine mixing contrast.
Fig. 7 is the high-efficient liquid phase chromatogram of Radix Aconiti, Radix Aconiti Kusnezoffii negative control solution.
Fig. 8 is the high-efficient liquid phase chromatogram of compound schizonepeta fumigation and washing agent sample.
Fig. 9 is the high-efficient liquid phase chromatogram of Radix Aconiti, Radix Aconiti Kusnezoffii.
Figure 10 is mesaconitine standard curve.
Figure 11 is hypaconitine standard curve.
Figure 12 is aconitine standard curve.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described further.
Embodiment 1
Treatment hemorrhoid Chinese medicine for outer use of the present invention is to be made up of the raw material of following parts by weight:
Herba Schizonepetae 120g Radix Saposhnikoviae 120g Herba speranskiae tuberculatae 300g
Radix Aconiti 90g Herba Potentillae Chinensis 300g Radix Aconiti Kusnezoffii 90g
Radix Sophorae Flavescentis 120g.
Preparation method is as follows:
First Herba Schizonepetae, Radix Aconiti, Radix Aconiti Kusnezoffii are ground into fine powder, then all the other raw materials are decocted with water 3 times, decocting time is respectively 2,2,1 hours, collecting decoction, filter, filtrate decompression is concentrated, dry, pulverize into fine powder, finally two kinds of fine powders are merged and mixed, granulate, be drying to obtain.
The discriminating of content:
The discrimination method concrete steps of Herba Schizonepetae are as follows:
Sample thief porphyrize, takes 6g, adds acetone 30ml supersound process 30 minutes, filter, and filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, as need testing solution; Get Herba Schizonepetae reference substance 1.0g, add acetone 20ml supersound process 30 minutes, filter, filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, product solution in contrast;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution and the each 5 μ l of reference substance solution, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, cyclohexane extraction-ethyl acetate taking volume ratio as 7:3 is developing solvent, launch, take out, dry, put under 365nm ultra-violet lamp and inspect; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, aobvious identical red fluorescence speckle.
The discrimination method concrete steps of Radix Saposhnikoviae are as follows:
Sample thief porphyrize, takes 6g, adds acetone 30ml supersound process 30 minutes, filter, and filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, as need testing solution; Get Radix Saposhnikoviae reference substance 1g, add acetone 20ml, supersound process 30 minutes, filters, filtrate evaporate to dryness, and residue adds ethanol 1ml to be made to dissolve, product solution in contrast;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution 10 μ l and reference substance solution 5 μ l, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, ethyl acetate-ethanol taking volume ratio as 4:1 is developing solvent, launch, take out, dry, put under 254nm ultra-violet lamp and inspect; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the fluorescence speckle of aobvious same color.
The discrimination method concrete steps of Radix Aconiti, Radix Aconiti Kusnezoffii are as follows:
Sample thief porphyrize, takes 5g, adds dehydrated alcohol 30ml supersound process 30 minutes, filter, dehydrated alcohol 5ml washing 2 times for filtering residue, merges with filtrate, evaporate to dryness, residue all dissolves with 10% hydrochloric acid solution 20ml, filters, filtrate is adjusted pH value 9～10 with ammonia, with ether jolting extraction 2 times, each 20ml, extracting solution low temperature evaporate to dryness, residue adds dehydrated alcohol 1ml to be made to dissolve, as need testing solution; Get aconitine reference substance, make every 1ml containing the solution of 1mg, product solution in contrast with dehydrated alcohol;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution and the each 10 μ l of reference substance solution, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, ether-ethyl acetate-ammonia taking volume ratio as 5:5:1 is developing solvent, launch, take out, dry, spray is with rare bismuth potassium iodide solution; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color.
The content assaying method concrete steps of matrine are as follows:
Reference " Chinese Pharmacopoeia " 2010 annex VI D high effective liquid chromatography for measuring:
(1) chromatographic condition and system suitability: with octadecylsilane chemically bonded silica be filler; Volume ratio is acetonitrile-0.05mol/L KH of 5:95:0.8:0.8
2pO
4-phosphoric acid-triethylamine is mobile phase; Detect wavelength 205nm;
(2) preparation of reference substance solution: get matrine reference substance, accurately weighed, add mobile phase and make the solution of every 1ml containing matrine 130 μ g, to obtain final product;
(3) preparation of need testing solution: sample thief porphyrize, get 0.25g, accurately weighed, put in tool plug conical flask, add strong ammonia solution 1ml and ether 30ml, close plug, hold over night, power is 250W, the frequency supersound process that is 33kHz 30 minutes, let cool, filter, container and residue divide 2 washings with ether 15ml, washing liquid and filtrate merge, evaporate to dryness, and residue adds the mutual-assistance of flowing and dissolves, be transferred in 25ml measuring bottle, be diluted to scale, shake up, filter, get subsequent filtrate, to obtain final product;
(4) algoscopy: accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.
The research that matrine content detects
(1) instrument and reagent
Instrument: the U.S. wears peace high performance liquid chromatograph, Scienhome Kromasil-C
18-5 μ 250 × 4.6mm chromatographic columns, P680 pump, UVD170 UV-detector, CHROMELEON data processing software, startorius BP211D electronic analytical balance (d=0.01mg).
Reagent: acetonitrile is chromatographically pure, water is redistilled water, all the other reagent are analytical pure.
Matrine reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, for assay for 0805-200005, about 20mg).
(2) selection of chromatographic condition
The selection of mobile phase: adopt the U.S. to wear peace high performance liquid chromatograph, P680 pump UVD170 UV-detector, CHROMELEON data processing software, flow velocity 1ml/ minute.Mobile phase is following system on probation once:
Mobile phase one: acetonitrile-dehydrated alcohol-3% phosphoric acid (80:10:10)
Result: do not detect the corresponding chromatographic peak of reference substance in 30 minutes.
Mobile phase two: acetonitrile-phosphate buffer-phosphoric acid-triethylamine (5:95:0.3:0.3)
Result: matrine retention time is 9.2 minutes, and reference substance product and sample all have hangover, and peak shape evaluation is general.
Mobile phase three: acetonitrile-0.05mol/L KH
2pO
4-phosphoric acid-triethylamine (5:95:0.8:0.8)
Result: matrine retention time is 8.9 minutes, the front lover of flowing of reference substance and sample peak shape, sample peak separates thoroughly.
According to result of the test, determine the mobile phase that mobile phase three is this method.
Detect determining of wavelength: according to " Chinese Pharmacopoeia " and reference, matrine mainly contains 205nm, 210nm, tri-absorbing wavelength of 220nm.For determining optimum absorb wavelength, this test is measured under three wavelength same reference substance and sample simultaneously, the results are shown in Table 1.
Table 1 detects wavelength selection result table
According to result of the test, matrine is in 205nm place peak area maximum, therefore determine that detecting wavelength is: 205nm.
(3) selection of sample extraction condition
Extract the selection of solvent: get this product under content uniformity item, porphyrize, gets about 0.25g, accurately weighed, triplicate, put respectively in tool plug conical flask, extract with following solvent respectively:
Method one, add strong ammonia solution 1ml and chloroform 30ml, close plug, hold over night, supersound process (power 250W, frequency 33kHz) 30 minutes, lets cool, filter, container and residue divide 2 washings with chloroform 15ml, and washing liquid and filtrate merge, evaporate to dryness, residue adds the mutual-assistance of flowing and dissolves, and is transferred in 25ml measuring bottle, be diluted to scale, shake up, filter, get subsequent filtrate, to obtain final product.
Method two, add strong ammonia solution 1ml and ether 30ml, close plug, hold over night, supersound process (power 250W, frequency 33kHz) 30 minutes, lets cool, filter, container and residue divide 2 washings with ether 15ml, and washing liquid and filtrate merge, evaporate to dryness, residue adds the mutual-assistance of flowing and dissolves, and is transferred in 25ml measuring bottle, be diluted to scale, shake up, filter, get subsequent filtrate, to obtain final product.
Method three, add strong ammonia solution 1ml and mobile phase 30ml, close plug, hold over night, supersound process (power 250W, frequency 33kHz) 30 minutes, lets cool, filter, container and residue divide 2 washings with mobile phase 15ml, and washing liquid and filtrate merge, steam to appropriate, be transferred in 25ml measuring bottle, add mobile phase and be diluted to scale, shake up, filter, get subsequent filtrate, to obtain final product.
Algoscopy: accurate reference substance solution and the above each need testing solution drawn, injection liquid chromatography respectively, measures peak area, the results are shown in Table 2.
Table 2 extracts solvent selection result table
Result shows: during taking ether as extraction solvent, sample size is the highest, therefore selective extraction solvent is ether.
The selection of extracting method: get this product under content uniformity item, porphyrize, gets about 0.25g, accurately weighed, in quadruplicate, put in tool plug conical flask, add respectively strong ammonia solution 1ml, ether 30ml, extract and prepare need testing solution with the following methods respectively, injection liquid chromatography respectively, measurement result is in table 3.
Table 3 extracting method selection result table
Result shows: sample is with after spending the night when supersound extraction, and matrine content is the highest, therefore selective extraction method is supersound extraction after spending the night.
Extract the selection of solvent load: get this product under content uniformity item, porphyrize, get about 0.25g, accurately weighed, triplicate, put in tool plug conical flask, add respectively strong ammonia solution 1ml, ether 20ml, 30ml, 40ml, the same legal system available test sample solution, inject respectively liquid phase chromatograph of liquid, measurement result is in table 4.
Table 4 solvent load selection result table
Result shows: when sample extracts with ether 30ml and 40ml, matrine content is suitable, for saving reagent, determines that the consumption of extraction solvent is 30ml.
The selection of extraction time: get this product under content uniformity item, porphyrize, gets about 0.25g, accurately weighed, triplicate, put respectively in tool plug conical flask, add strong ammonia solution 1ml and ether 30ml, close plug, hold over night, respectively supersound process (power 250W, frequency 33kHz) 20 minutes, 30 minutes, 40 minutes, the same legal system available test sample solution, injection liquid chromatography is surveyed its content respectively, and measurement result is in table 5.
Table 5 extraction time selection result table
Result shows: ultrasonic 30 minutes time, effective ingredient has extracted completely, and ultrasonic 40 minutes time, sample size is without significant change, therefore determine that the sample ultrasonic time is 30 minutes.
(4) range of linearity is investigated: get matrine reference substance appropriate, and accurately weighed, add mobile phase and make the contrast solution of every 1ml containing matrine 130 μ g, accurate 20 μ l, 15 μ l, 10 μ l, 5 μ l, the 2 μ l sample introductions drawn, measure its peak area respectively.The results are shown in Table 6.With peak area (Y), sample size (X) is returned, obtain standard curve equation.Obtaining matrine standard curve equation is: Y=29.969X-0.6211(r=0.9998), matrine standard curve is shown in Fig. 1.Above result shows that matrine is within the scope of 0.26 μ g～2.60 μ g, and peak area value and sample size have good linear relationship.
Table 6 matrine reference substance measurement result table
(5) negative control test: get the not negative pilot sample 0.25g containing Radix Sophorae Flavescentis, Sophora flavescens fine powder 0.04g, after the same method is processed, make respectively negative controls and Sophora flavescens solution, get respectively reference substance solution, negative controls, Sophora flavescens solution, compound schizonepeta fumigation and washing agent need testing solution and respectively enter 20 μ l injection liquid chromatographies, record chromatogram, result is as Fig. 2, Fig. 3, Fig. 4, Fig. 5.
Result shows: in negative control collection of illustrative plates, matrine peak is noiseless.
(6) precision test: draw matrine reference substance solution (26 μ g/ml) and 050501 batch of compound schizonepeta fumigation and washing agent same sample test liquid and repeat respectively sample introduction 6 times, each each 20 μ l, record respectively both peak areas and calculate RSD, the results are shown in Table 7.
Table 7 Precision test result
Result shows: matrine reference substance peak area RSD is 0.19%, and test sample peak area RSD is 1.83%, and the precision of reference substance and test sample is good.
(7) stability test: get 050501 batch with a need testing solution, respectively at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, sample introduction.The results are shown in Table 8.
Table 8 sample stability result of the test
Result shows: test sample matrine peak area RSD in 8 hours is 0.44%, and need testing solution is good at 8 hours internal stabilities.
(8) repeatability test: get 5 parts of 050501 batch samples, press respectively the preparation of test sample preparation method, measure peak area, the results are shown in Table 9.
Table 9 reproducible test results
Result shows: the average content of 5 parts of test samples is 2.72mg/g, and RSD is 1.01%, and the repeatability of test method is good.
(9) recovery test: (adopting application of sample absorption method) precision takes 050501 crowd of (known matrine content is 2.72mg/g) sample 0.12g, gets altogether 5 parts, adds respectively identical matrine reference substance, measures total content, calculate recovery rate.The response rate=(measuring in total amount-sample)/add sterling amount × 100%.The results are shown in Table 10.
Table 10 (+)-Matrine hydrochloride recovery test result
Result shows: matrine average recovery rate is that 98.8%, RSD is 0.14%, and average recovery is good.
(10) sample determination: get compound schizonepeta fumigation and washing agent sample, measure by the inventive method, matrine content is in table 11.
Table 11 compound schizonepeta fumigation and washing agent determination of matrine result
Result shows: content is all stabilized in as more than 1.8mg/g, proves that this content assaying method is stable, can well control the quality of product.
The content assaying method concrete steps of aconite alkaloids are as follows:
With reference to " Chinese Pharmacopoeia " 2010 editions annex VI D high effective liquid chromatography for measuring
(1) chromatographic condition and system suitability: with octadecylsilane chemically bonded silica be filler; Volume ratio is that acetonitrile-oxolane-0.1mol/L Spirit of Mindererus. (every 1000ml adds glacial acetic acid 0.5ml) of 55:33:312 is mobile phase; Detection wavelength is 230nm; Number of theoretical plate calculates and should be not less than 4000 by aconitine peak;
(2) preparation of reference substance solution: get aconitine, hypaconitine, mesaconitine reference substance, accurately weighed, the mixed solution that adds volume ratio and be hydrochloric acid-methanol of 1:100 is made the mixed solution of every 1ml containing aconitine, hypaconitine, the each 30 μ g of mesaconitine, to obtain final product;
(3) preparation of need testing solution: sample thief porphyrize, get 1g, accurately weighed, put in tool plug conical flask, it is the mixed solution 50ml of hydrochloric acid-methanol of 1:100 that precision adds volume ratio, weighed weight, water temperature is controlled at 20 DEG C, and power is 250W, the frequency supersound process that is 50kHz 40 minutes, lets cool, weighed weight again, the mixed solution of the hydrochloric acid-methanol that is 1:100 by volume ratio is supplied the weight of less loss, shakes up, and filters, get subsequent filtrate, to obtain final product;
(4) algoscopy: accurate reference substance solution and the each 20ul of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.
The research of aconite alkaloids content detection
(1) instrument and reagent
Instrument: the U.S. wears peace high performance liquid chromatograph (P680 pump, UVD170U UV-detector, C18 post, CHROMELEON data processing software); Startorius BP211D electronic analytical balance (d=0.01mg), startorius BS124S electronic analytical balance (d=0.1mg); BENCHTOP CLFANERS HS3120 ultrasonator.
Reagent: acetonitrile (HPLC), methanol (HPLC), methanol (AR), glacial acetic acid (AR), oxolane (AR), ammonium acetate (AR), hydrochloric acid (AR), water is redistilled water.
Aconitine (lot number: 110720-200410, Chinese pharmaceutical biological product is identified institute)
Hypaconitine (lot number: 798-9202, Chinese pharmaceutical biological product is identified institute)
Mesaconitine (lot number: 110799-200404, Chinese pharmaceutical biological product is identified institute)
(2) selection of method
Measure according to method in the present invention, result of the test is that aconitine, hypaconitine, mesaconitine peak separating degree are good, and the number of plates is all greater than 4000, and method is feasible.
(3) negative control test: get respectively compound schizonepeta fumigation and washing agent 100401 batch samples, containing the negative pilot sample 1g of Radix Aconiti, Radix Aconiti Kusnezoffii, not accurately weighed, prepare with need testing solution, after same treatment, make compound schizonepeta fumigation and washing agent need testing solution, negative control solution; Get Radix Aconiti medical material 0.221g, Radix Aconiti Kusnezoffii 0.221g, with need testing solution preparation, after same treatment, obtains Radix Aconiti, Radix Aconiti Kusnezoffii solution; Get respectively aconitine, hypaconitine, mesaconitine mixing reference substance solution, Radix Aconiti, the two negative control solutions of Radix Aconiti Kusnezoffii, compound schizonepeta fumigation and washing agent need testing solution, Radix Aconiti, the each 20 μ l sample introductions of Radix Aconiti Kusnezoffii solution, record chromatogram.Result of the test is shown in Fig. 6, Fig. 7, Fig. 8, Fig. 9.
Result shows: at negative control collection of illustrative plates mesaconitine, hypaconitine, mesaconitine is all noiseless.
(4) replica test: get 100401 batch samples under content uniformity item, porphyrize, gets 6 parts, every part of about 1g, accurately weighed, to put in tool plug conical flask, it is the mixed solution 50ml of hydrochloric acid-methanol of 1:100 that precision adds volume ratio, weighed weight, supersound process (power 250w, frequency 50kHz; Water temperature is controlled at 20 DEG C) 40 minutes, let cool, more weighed weight, the mixed solution of the hydrochloric acid-methanol that is 1:100 by volume ratio is supplied the weight of less loss, shakes up, and filters, and gets subsequent filtrate, to obtain final product.Result of the test is in table 12.
Table 12 replica test result
Result shows: sample mesaconitine, hypaconitine, mesaconitine total content RSD are 0.20%, and method repeatability is good.
(5) precision test in the middle of: get 100401 batch samples under content uniformity item, according to the preparation method processing sample of need testing solution, at same date not, by different analysts, test respectively at different instruments, wherein sample 1,2 is same date not, sample 3,4 is different operating personnel, and sample 5,6 is different Instrument measurings.Result of the test is in table 13.
Precision test result in the middle of table 13
Result shows: in the middle of sample mesaconitine, hypaconitine, mesaconitine total content, precision RSD is 0.30%, and middle precision is good.
(6) range of linearity is investigated: get mesaconitine, hypaconitine, aconitine reference substance appropriate, accurately weighed, the mixed solution that adds volume ratio and be hydrochloric acid-methanol of 1:100 is made the solution of every 1ml containing mesaconitine 36.8 μ g, hypaconitine 39.6 μ g, aconitine 36 μ g, must contrast one, the accurate 5ml of absorption contrasts one and moves in 10ml volumetric flask, be settled to 10ml by mobile phase, must contrast two, take turns doing gradient dilution, must contrast three, contrast four, contrast five, the accurate each 20 μ l sample introductions of above-mentioned reference substance solution of drawing, measure its peak area respectively.The results are shown in Table 14, table 15, table 16.With peak area (y), sample size is (x) returned, obtain mesaconitine standard curve equation: y=17.0240x+0.0157(r=0.99995), hypaconitine normal equation is: y=16.3203x-0.0113(r=0.99993), aconitine normal equation is y=16.5254x-0.0232(r=0.99991) above result shows that mesaconitine is within the scope of 0.046 μ g～0.736 μ g, hypaconitine is within the scope of 0.0495 μ g～0.792 μ g, aconitine is within the scope of 0.045 μ g～0.72 μ g, and peak area value and sample size have good linear relationship.Result of the test is shown in Figure 10, Figure 11, Figure 12.
Table 14 mesaconitine reference substance measurement result
Table 15 hypaconitine reference substance measurement result
Table 16 aconitine reference substance measurement result
(7) recovery test: (adopting application of sample absorption method) precision takes 100401 batches of (known content mesaconitine 2.61mg/ bags, be 0.261mg/g, hypaconitine 3.09mg/ bag, be 0.309mg/g, aconitine 2.40mg/ bag, i.e. 0.240mg/g) sample 0.5g, nominal is got 5 parts, add respectively identical mesaconitine, hypaconitine, aconitine mixing reference substance, measure total content, calculate recovery rate.The response rate=(measuring in total amount-sample)/add sterling amount.The results are shown in Table 17.
Table 17 application of sample recovery test result
Result shows: the average recovery rate of mesaconitine is that 100.14%, RSD is 0.61%, and the average recovery rate of hypaconitine is that 99.67%, RSD is 0.52%, and the average recovery rate of aconitine is that 99.46%, RSD is 0.81%, and average recovery is good.
(8) sample determination: get ten batch samples, measure by the method for working out, content is in table 18.
Table 1810 batch sample assay result
By finding out in above 10 batches of compound schizonepeta fumigation and washing agent sample determinations, what mesaconitine+hypaconitine+mesaconitine total content was minimum is 090501 batch, and content is 4.81mg/ bag, and what content was the highest is 110101 batches, is 12.22mg/ bag.Final definite diester-type alkaloids is with aconitine (C
34h
47nO
11), hypaconitine (C
34h
45nO
10), mesaconitine (C
33h
45nO
11) total amount and every bag be 3.0mg～14.5mg, each use amount is one bag or pastille scope for each use amount.
Claims (1)
1. treat the detection method of hemorrhoid Chinese medicine for outer use for one kind, comprise the discriminating of content and the assay that contains composition, it is characterized in that: the discriminating of content is that thin layer chromatography is differentiated Herba Schizonepetae, Radix Saposhnikoviae, Radix Aconiti, Radix Aconiti Kusnezoffii composition, and the assay that contains composition is the content of high effective liquid chromatography for measuring matrine, aconite alkaloids;
The discrimination method concrete steps of described Herba Schizonepetae are as follows:
Sample thief porphyrize, takes 6g, adds acetone 30ml supersound process 30 minutes, filter, and filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, as need testing solution; Get Herba Schizonepetae reference substance 1.0g, add acetone 20ml supersound process 30 minutes, filter, filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, product solution in contrast;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution and the each 5 μ l of reference substance solution, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, cyclohexane extraction-ethyl acetate taking volume ratio as 7:3 is developing solvent, launch, take out, dry, put under 365nm ultra-violet lamp and inspect; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, aobvious identical red fluorescence speckle;
The discrimination method concrete steps of described Radix Saposhnikoviae are as follows:
Sample thief porphyrize, takes 6g, adds acetone 30ml supersound process 30 minutes, filter, and filtrate evaporate to dryness, residue adds ethanol 1ml to be made to dissolve, as need testing solution; Get Radix Saposhnikoviae reference substance 1g, add acetone 20ml, supersound process 30 minutes, filters, filtrate evaporate to dryness, and residue adds ethanol 1ml to be made to dissolve, product solution in contrast;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution 10 μ l and reference substance solution 5 μ l, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, ethyl acetate-ethanol taking volume ratio as 4:1 is developing solvent, launch, take out, dry, put under 254nm ultra-violet lamp and inspect; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the fluorescence speckle of aobvious same color;
Described Radix Aconiti, the discrimination method concrete steps of Radix Aconiti Kusnezoffii are as follows:
Sample thief porphyrize, takes 5g, adds dehydrated alcohol 30ml supersound process 30 minutes, filter, dehydrated alcohol 5ml washing 2 times for filtering residue, merges with filtrate, evaporate to dryness, residue all dissolves with 10% hydrochloric acid solution 20ml, filters, filtrate is used ammonia adjust pH 9～10, with ether jolting extraction 2 times, each 20ml, extracting solution low temperature evaporate to dryness, residue adds dehydrated alcohol 1ml to be made to dissolve, as need testing solution; Get aconitine reference substance, make every 1ml containing the solution of 1mg, product solution in contrast with dehydrated alcohol;
Detect with reference to " Chinese Pharmacopoeia " version annex VI B thin layer chromatography in 2010, draw need testing solution and the each 10 μ l of reference substance solution, put respectively on the same silica gel g thin-layer plate taking sodium carboxymethyl cellulose as adhesive, ether-ethyl acetate-ammonia taking volume ratio as 5:5:1 is developing solvent, launch, take out, dry, spray is with rare bismuth potassium iodide solution; In test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle of aobvious same color;
The content assaying method concrete steps of described matrine are as follows:
Reference " Chinese Pharmacopoeia " 2010 annex VI D high effective liquid chromatography for measuring:
(1) chromatographic condition and system suitability: with octadecylsilane chemically bonded silica be filler; Volume ratio is acetonitrile-0.05mol/L KH of 5:95:0.8:0.8
2pO
4-phosphoric acid-triethylamine is mobile phase; Detect wavelength 205nm;
(2) preparation of reference substance solution: get matrine reference substance, accurately weighed, add mobile phase and make the solution of every 1ml containing matrine 130 μ g, to obtain final product;
(3) preparation of need testing solution: sample thief porphyrize, get 0.25g, accurately weighed, put in tool plug conical flask, add strong ammonia solution 1ml and ether 30ml, close plug, hold over night, power is 250W, the frequency supersound process that is 33kHz 30 minutes, let cool, filter, container and residue divide 2 washings with ether 15ml, washing liquid and filtrate merge, evaporate to dryness, and residue adds the mutual-assistance of flowing and dissolves, be transferred in 25ml measuring bottle, be diluted to scale, shake up, filter, get subsequent filtrate, to obtain final product;
(4) algoscopy: accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product;
The content assaying method concrete steps of described aconite alkaloids are as follows:
Reference " Chinese Pharmacopoeia " 2010 annex VI D high effective liquid chromatography for measuring:
(1) chromatographic condition and system suitability: with octadecylsilane chemically bonded silica be filler; Volume ratio is acetonitrile-oxolane-0.1mol/L Spirit of Mindererus. of 55:33:312, and in every 1000ml0.1mol/L Spirit of Mindererus., adding glacial acetic acid 0.5ml is mobile phase; Detection wavelength is 230nm; Number of theoretical plate calculates and should be not less than 4000 by aconitine peak;
(2) preparation of reference substance solution: get aconitine, hypaconitine, mesaconitine reference substance, accurately weighed, the mixed solution that adds volume ratio and be hydrochloric acid-methanol of 1:100 is made the mixed solution of every 1ml containing aconitine, hypaconitine, the each 30 μ g of mesaconitine, to obtain final product;
(3) preparation of need testing solution: sample thief porphyrize, get 1g, accurately weighed, put in tool plug conical flask, it is the mixed solution 50ml of hydrochloric acid-methanol of 1:100 that precision adds volume ratio, weighed weight, water temperature is controlled at 20 DEG C, and power is 250W, the frequency supersound process that is 50kHz 40 minutes, lets cool, weighed weight again, the mixed solution of the hydrochloric acid-methanol that is 1:100 by volume ratio is supplied the weight of less loss, shakes up, and filters, get subsequent filtrate, to obtain final product;
(4) algoscopy: accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures, and to obtain final product.
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