CN103575849A - Quality test method for corydalis saxicola bunting total alkali - Google Patents
Quality test method for corydalis saxicola bunting total alkali Download PDFInfo
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Abstract
The invention relates to a quality test method for corydalis saxicola bunting total alkali. The method comprises the following steps: identifying a thin layer of an effective part, inspecting moisture, residue on ignition, heavy metal and arsenic salt, and measuring the content of total alkaloids by adopting an ultraviolet spectrophotometry and measuring the content of dehydrogenase crushed leaves corydaline, dehydrogenation A Kaweiting, jateorhizine, dehydrocavidine, palmatine chloride and berberine hydrochloride. The method has strong specificity and good reproducibility. By utilizing the quality test method, the quality of the corydalis saxicola bunting total alkali can be effectively controlled, and a product can be ensured to be safe, efficient, and controllable in quality.
Description
Technical field
The invention belongs to Analysis of Chinese Traditional Medicine field, relate to a kind of quality determining method of effective ingredient in Chinese, be specifically related to a kind of quality determining method of corydalis saxicola total alkaloid.
Background technology
Meadowrueleaf corydalis root is the herb of the raw yellow violet Corydalis saxicola Bunting of bloodroot stone, is the Chinese medicinal herbs of ,Gui high and cold mountain area, Guizhou Province preciousness.This product bitter, cool in nature, tool eliminating dampness and heat, eliminating stasis to subdue swelling effect.For sore furuncle poison, hepatitis, cirrhosis, liver cancer.
Few to the research of the chemical composition of meadowrueleaf corydalis root up to now, the now separated compound identifying of report is mainly alkaloid, not yet has the report of other compounds.1980, Ke Min jade-like stone was studied the chemical composition of meadowrueleaf corydalis root the earliest, application pH gradient separations, at strong basicity position, get water-soluble quaternary ammonium base Alkaloid jamaicin (berberine) and deydrokaividing (Corydalis Saxicolae, dehydrocavidine).K. H. Janbaz equals report in 2003, and water-soluble quaternary ammonium base compounds, as jamaicin, has liver injury protection effect.
In meadowrueleaf corydalis root, main effective constituent is to take the alkaloid compound that deydrokaividing is representative, and this compounds has potential anti-HBV activity and liver protective effect.
Through retrieval, the patent about meadowrueleaf corydalis root mainly concentrates in the alkaloidal preparation technology of meadowrueleaf corydalis root at present, there is no the Patents about quality control.Chinese medicine for a long time, effectively control by difficult quality, and the quality of effectively controlling tcm product is a great problem, and the method for available technology adopting is imperfect, and minority specific aim is not strong, uses these methods to be difficult to reach the object of real control quality.Above corydalis saxicola bunting total alkali, owing to lacking the method for quality control of system, is difficult to control its quality, and consequently normal clinical application and curative effect cannot guarantee.
In the quality standard research of Related Rocks coptis, the quality standard of listing kind meadowrueleaf corydalis root parenteral solution is to adopt the qualitative discriminating deydrokaividing of thin-layer chromatography and Berberine hydrochloride, adopts the content of high effective liquid chromatography for measuring deydrokaividing at present.The meadowrueleaf corydalis root sheet of bibliographical information and meadowrueleaf corydalis root rectal plug are all the quality standards of setting up with the same method of meadowrueleaf corydalis root parenteral solution adopting.The present invention is directed to the deficiencies in the prior art, adopt new means to control the quality of corydalis saxicola bunting total alkali, particularly adopt the content of Multiple components in this medicine of hplc simultaneous determination, solve a difficult problem for corydalis saxicola bunting total alkali quality control, the invention provides a kind of method of quality control of practicality.The method provides thin layer discriminating, the inspections such as moisture, residue on ignition, content of beary metal, the method of quality control such as content of Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride, Berberine hydrochloride composition in corydalis saxicola bunting total alkali and high effective liquid chromatography for measuring medicine in determined by ultraviolet spectrophotometry medicine, quality standard after raising can be controlled the quality of corydalis saxicola bunting total alkali better, method of quality control of the present invention, there is stronger specificity and good reappearance, really embody Chinese medicine safe and effective, quality controllable.
Summary of the invention
The object of the present invention is to provide a kind of method of quality control of meadowrueleaf corydalis root.In order to reach this object, adopt following technical scheme:
A quality determining method for corydalis saxicola bunting total alkali, the method comprises the following steps:
(1) take meadowrueleaf corydalis root as control medicinal material, take deydrokaividing, palmatin hydrochloride, Berberine hydrochloride is reference substance, and corydalis saxicola bunting total alkali is carried out to thin layer discriminating;
(2) moisture of corydalis saxicola bunting total alkali, residue on ignition, heavy metal and arsenic salt are checked;
(3) adopt the content of total alkaloids in determined by ultraviolet spectrophotometry corydalis saxicola bunting total alkali;
(4) adopt the content of Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride and Berberine hydrochloride in hplc simultaneous determination corydalis saxicola bunting total alkali.
The quality determining method of above-mentioned corydalis saxicola bunting total alkali, the thin-layer identification method step of corydalis saxicola bunting total alkali is:
A. the preparation of need testing solution: get this product 10mg, add 25ml methyl alcohol to dissolve, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution;
B. the preparation of control medicinal material solution: get meadowrueleaf corydalis root control medicinal material 0.1g, add 5ml methyl alcohol, ultrasonic processing 30 minutes, filters, and filtrate is medicinal material solution in contrast;
C. the preparation of reference substance solution: get deydrokaividing, palmatin hydrochloride, Berberine hydrochloride reference substance, add methyl alcohol and make every 1ml containing the mixed liquor of deydrokaividing 0.2mg, palmatin hydrochloride 0.2mg, Berberine hydrochloride 0.1mg, as mixing reference substance solution;
D. thin layer condition and result: the thin-layered chromatography of recording according to appendix VI B of Chinese Pharmacopoeia version in 2010 is tested, draw each 5 μ l of above-mentioned three kinds of solution, point be take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder in same, take ratio as 9: 2: 5: ethyl acetate-methylene chloride-methanol-strong ammonia solution of 3, and normal butyl alcohol-glacial acetic acid-water that ratio is 7: 1: 2 is developping agent, launch, take out, dry up, putting wavelength is to inspect under 365nm ultraviolet lamp, in test sample chromatogram, with reference substance and the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.
The quality determining method of above-mentioned corydalis saxicola bunting total alkali, the inspection method step of corydalis saxicola bunting total alkali moisture, residue on ignition, heavy metal and arsenic salt is:
A. the aquametry of recording according to appendix IX H first method of Chinese Pharmacopoeia version in 2010 is measured the moisture of corydalis saxicola bunting total alkali, and in corydalis saxicola bunting total alkali, moisture must not surpass 5.0%;
B. the residue on ignition determination method of recording according to appendix IX J of Chinese Pharmacopoeia version in 2010 is measured the residue on ignition of corydalis saxicola bunting total alkali, and in corydalis saxicola bunting total alkali, residue on ignition must not cross 2.0%;
C. the heavy metal inspection method that appendix IX E of Chinese Pharmacopoeia version in 2010 the second method is recorded is measured, the ancient Cai Shi method of first method that the inspection of arsenic salt is recorded by appendix IX F of Chinese Pharmacopoeia version in 2010 is measured, in corydalis saxicola bunting total alkali, heavy metal must not cross 10/1000000ths, and arsenic salt must not cross 2/1000000ths.
The quality determining method of above-mentioned corydalis saxicola bunting total alkali, in determined by ultraviolet spectrophotometry corydalis saxicola bunting total alkali, the content step of total alkaloids is:
A. the preparation of the reference substance solution about 10mg of deydrokaividing reference substance of phosphorus pentoxide dried overnight that learns from else's experience, accurately weighed, puts in 100ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up, as stock solution, standby;
B. the preparation precision of typical curve measures reference substance stock solution 0.3ml, 0.4m1, and 0.5ml, 0.6ml, 0.8ml, 1.0ml, puts respectively in 10ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up; Take 1% methanol hydrochloride solution as blank, and the UV-VIS spectrophotometry of recording according to appendix V A of Chinese Pharmacopoeia version in 2010, measures absorbance log at 347nm wavelength place, take absorbance log as ordinate, concentration be horizontal ordinate drawing standard curve;
C. the about 25mg of this product is got in the preparation of need testing solution, accurately weighed, puts in 50ml volumetric flask, adds 1% methanol hydrochloride solution to scale, as sample stock solution, standby; The accurate sample stock solution 1.0ml that draws puts in 50ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up, and obtains;
D. determination method: get need testing solution, take 1% methanol hydrochloride solution as blank, measure absorbance log in accordance with the law, the amount of reading deydrokaividing in need testing solution from typical curve, calculates, and obtains.This product in deydrokaividing C21H23O4N, must not be less than 50.0% containing total alkaloids.
The quality determining method of above-mentioned corydalis saxicola bunting total alkali, in hplc simultaneous determination corydalis saxicola bunting total alkali, the content step of Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride and Berberine hydrochloride is:
A. chromatographic condition and system suitability adopt Hanbon ODS-2C18, specification is 250mm * 4.6mm, the chromatographic column of 5 μ m, acetonitrile-0.01mol/L potassium dihydrogen phosphate aqueous solution that the ratio of take is 18.5: 81.5 is mobile phase, 30 ℃ of column temperatures, detect wavelength 347nm, flow velocity 1.0ml/min, sample size 10 μ l, sampling time 50min;
B. it is appropriate that the preparation precision of reference substance solution takes Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride, Berberine hydrochloride, add methyl alcohol and make respectively single standard items storing solution, it is appropriate that precision is got each storing solution respectively, be mixed with hybrid standard product storing solution, the mass concentration of above 6 compounds in hybrid standard product storing solution is respectively 0.0238,0.0889,0.0134,0.098,0.06292,0.0174mg/mL;
C. the about 25mg of this product is got in the need testing solution preparation of preparing of need testing solution, accurately weighed, puts in 50ml measuring bottle, adds 1% hydrochloric acid methanol to scale, shakes up, and crosses 0.45 μ m diameter miillpore filter, gets subsequent filtrate, obtains;
D. determination method: accurate respectively 10 μ l of reference substance and need testing solution that draw respectively, injection liquid chromatography, measure, obtain, this product must not be less than 32.0% containing Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, dehydrocavidine, palmatin hydrochloride, Berberine hydrochloride total amount.
Beneficial effect:
The present invention has proposed method of quality control to corydalis saxicola bunting total alkali, the method provides the content of corydalis saxicola bunting total alkali and Scoulerine in discriminating, inspection, ultraviolet spectrophotometry and hplc simultaneous determination medicine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride, Berberine hydrochloride, quality standard after raising can be controlled the quality of meadowrueleaf corydalis root better, method of quality control of the present invention, there is stronger specificity and good reappearance, really embody Chinese medicine safe and effective, quality controllable.
Accompanying drawing explanation
Fig. 1 be test sample of the present invention ethyl acetate-methylene chloride-methanol-strong ammonia solution (9: 2: 5: the 3) thin-layer chromatogram under unfolding condition, 20071018), sample 1(lot number 20071014), sample 1(lot number wherein band is followed successively by from left to right: mix reference substance, meadowrueleaf corydalis root control medicinal material, sample 1(lot number::: 20071022);
20071018), sample 1(lot number 20071014), sample 1(lot number Fig. 2 is the thin-layer chromatogram of test sample of the present invention under normal butyl alcohol-glacial acetic acid-water (7: 1: 2) unfolding condition, and wherein band is followed successively by from left to right: mix reference substance, meadowrueleaf corydalis root control medicinal material, sample 1(lot number::: 20071022);
Fig. 3 is Scoulerine of the present invention, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride, (in figure, 1 is Scoulerine to Berberine hydrochloride mixing reference substance liquid chromatogram, 2 is dehydrogenation first Ka Weiting, 3 is jateorrhizine, 4 is deydrokaividing, 5 is palmatin hydrochloride, and 6 is Berberine hydrochloride);
Fig. 4 is corydalis saxicola bunting total alkali test sample of the present invention (lot number: 20071014) liquid chromatogram (the same Fig. 3 of label).
Embodiment
The method for measuring simultaneously of sesquiterpenoids of the present invention and curcumin composition is the optimal case obtaining through a large amount of shaker tests, and in conjunction with embodiment, the present invention is further described as follows:
1. corydalis saxicola bunting total alkali thin layer is differentiated:
1.1 instruments, reagent and reagent
FA1104 type ten thousand/electronic analytical balance (Shanghai balance equipment factory); Uv analyzer WD-9403C type (Beijing Liuyi Instrument Factory); Water-bath (Gongyi City Ying Yu Yu Hua instrument plant); Silica G, H thin layer plate (Haiyang Chemical Plant, Qingdao); Corydalis saxicola bunting total alkali (lot number: 20071014,20071018,20071022); Deydrokaividing reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, for assay, lot number: 111667-200601); Palmatin hydrochloride reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, for assay, lot number: 110732-200506); Berberine hydrochloride reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, for assay, lot number: 110713-200508).Normal butyl alcohol is for analyzing pure (Shanghai sincere chemical reagent company limited, lot number: 20080227); Ethyl acetate is for analyzing pure (Chemical Reagent Co., Ltd., Sinopharm Group, lot number: T20090406); Ammoniacal liquor is for analyzing pure (Nanjing Chemistry Reagent Co., Ltd., lot number: 080920759); Glacial acetic acid is for analyzing pure (Chemical Reagent Co., Ltd., Sinopharm Group, lot number: T20090305); The concentrated sulphuric acid is for analyzing pure (Shanghai Jiu Yi chemical reagent company limited, lot number: 091022); Ethanol is for analyzing pure (Nanjing Chemistry Reagent Co., Ltd., lot number: 09081310871); Ether is for analyzing pure (pilot scale chemical corp, Shanghai, lot number: 20100415); Normal hexane is for analyzing pure (Shishewei Chemical Co., Ltd., Shanghai, lot number: 0904102); Methylene chloride is for analyzing pure (Shanghai Ling Feng chemical reagent company limited, lot number: 042101); Water is dual distilled water.
1.2 identification experiment methods
Corydalis saxicola total alkaloid thin-layer chromatography discrimination condition is: get this product 10mg, add 25ml methyl alcohol to dissolve, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution.Separately get meadowrueleaf corydalis root control medicinal material 0.1g, add 5ml methyl alcohol, ultrasonic processing 30 minutes, filters, and filtrate is medicinal material solution in contrast.Get again deydrokaividing reference substance, palmatin hydrochloride reference substance, Berberine hydrochloride reference substance, add methyl alcohol and make every 1ml containing the mixed liquor of deydrokaividing 0.2mg, palmatin hydrochloride 0.2mg, Berberine hydrochloride 0.1mg, as mixing reference substance solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2010), test, draw each 5 μ l of above-mentioned three kinds of solution, point be take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder in same, take ethyl acetate-methylene chloride-methanol-strong ammonia solution (9: 2: 5: 3) and normal butyl alcohol-glacial acetic acid-water (7: 1: 2) be developping agent, launch, take out, dry up, put under ultraviolet lamp (365nm) and inspect.In test sample chromatogram, with reference substance and the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.The results are shown in Figure 1,2.
2 check:
With reference to < < Chinese Pharmacopoeia > > and correlation technique requirement, the moisture of this product, residue on ignition, heavy metal and arsenic salt are checked and measured.
2.1 moisture are measured according to aquametry (appendix IX H first method of version < < Chinese Pharmacopoeia > > in 2010), the results are shown in Table 1.
Table 1 corydalis saxicola total alkaloid determination of moisture value
Result shows, three batches of pilot scale sample moisture all do not cross 5.0%, therefore moisture must not cross 5.0% in regulation corydalis saxicola bunting total alkali.
2.2 residue on ignition residue on ignition inspections are measured by appendix IX J of version < < Chinese Pharmacopoeia > > in 2010, the results are shown in Table 2.
Table 2 corydalis saxicola total alkaloid residue on ignition measured value
Result shows, test agent residue on ignition does not all cross 2.0% in three batches, therefore residue on ignition must not cross 2.0% in regulation corydalis saxicola bunting total alkali.
The inspection of 2.3 heavy metals and arsenic salt heavy metal is measured by appendix IX E second method of < < Chinese Pharmacopoeia > > version in 2010, the inspection of arsenic salt is measured by a < < Chinese Pharmacopoeia > > 2010 appendix IX F first method of version (ancient Cai Shi method), and the check result of three batch samples is in Table table 3, table 4.
Table 3 three batch sample heavy metal check results
Table 4 three batch sample arsenic salt check results
By the detection to three batch sample heavy metals and arsenic salt, result all meets the requirement of drug safety technical manual, therefore heavy metal must not cross 10/1000000ths in regulation corydalis saxicola bunting total alkali, arsenic salt must not cross 2/1000000ths.
3 assays
3.1 instruments and reagent
Agillent1100 high performance liquid chromatograph, comprises quaternary pump, automatic sampler, column oven, vacuum degassing machine, UV-detector; Chromatographic column is Agilent XDB C18 (250mm * 4.6mm, 5 μ m); Shimadzu UV2550 ultraviolet-visible spectrophotometer, 752 type ultraviolet-visible spectrophotometers.Acetonitrile is chromatographically pure (TEDIA), and water is redistilled water, KH
2pO
4, phosphoric acid, hydrochloric acid, methyl alcohol, be all analyze pure.Corydalis saxicola bunting total alkali extract (lot number: 20071014,20071018,20071022).Deydrokaividing reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, for assay, lot number: 111667-200601), palmatin hydrochloride reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, for assay, lot number: 110732-200506), Berberine hydrochloride reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, for assay, lot number: 110713-200508).Scoulerine, dehydrogenation first Ka Weiting, the self-control of jateorrhizine reference substance, warp
1h-NMR,
13c-NMR, UV, the methods such as MS are determined its structure, HPLC normalization method determines that its purity is all greater than 98%.
3.2 corydalis saxicola bunting total alkalis are measured
According to ultraviolet-spectrophotometric method (appendix VA of < < Chinese Pharmacopoeia > > version in 2010), measure and set up the ultraviolet spectrophotometry content assaying method of corydalis saxicola bunting total alkali.
3.2.1 measure the selection of wavelength
The preparation of reference substance solution: precision takes the about 10.06mg of deydrokaividing reference substance, puts in 100ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up, as stock solution, standby.
Deydrokaividing reference substance and this product are mixed with respectively to the solution of 20 μ g/ml with 1% methanol hydrochloride solution, 1% methanol hydrochloride solution is blank, in the interscan of 200～800nm wavelength coverage, reference substance and sample all have smooth absorption at 347nm place, this is consistent with the ultraviolet-visible spectrogram of medicinal material, therefore select 347nm as the wavelength of assay.
3.2.2 the investigation of the range of linearity
Precision measures reference substance stock solution 0.3ml, 0.4ml, and 0.5ml, 0.6ml, 0.8ml, 1.0ml, puts respectively in 10ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up; Take 1% methanol hydrochloride solution as blank, measure absorbance at 347nm wavelength place, take absorbance as ordinate, concentration is horizontal ordinate, drawing standard curve, and regression equation is: Y=0.0704X-0.0088, r=0.9997.Result shows that corydalis saxicola total alkaloid is good linear relationship within the scope of 3.018 μ g/ml～10.06 μ g/ml, in Table 5.
Table 5 linear test result
3.2.3 precision test
Get the reference substance solution that concentration is 10.06 μ g/ml, replication 6 times, the results are shown in Table 6.
Table 6 Precision test result
The RSD of the corydalis saxicola bunting total alkali ultraviolet absorptivity of measuring for 6 times is 0.27%, and precision meets content assaying method and learns the requirement of investigating.
3.2.4 replica test
Get with a collection of corydalis saxicola bunting total alkali powder (lot number: 20071014) 6 parts, accurately weighed, put respectively in 50ml measuring bottle, add 1% methanol hydrochloride solution and dissolve and constant volume, precision measures subsequent filtrate 1.0mI and puts in 50m1 measuring bottle, adds 1% methanol hydrochloride solution to scale, shake up, in different time, measure ultraviolet absorptivity successively, calibration curve method calculates content, the results are shown in Table 7.
Table 7 replica test result
Conclusion: the repeatability of sample meets the requirement that total alkali mensuration methodology is investigated.
3.2.5 stability test
Get No. 1 need testing solution in this product replica test, in different time, measure ultraviolet absorptivity successively, the results are shown in Table 8.
Table 8 stability test result
Result shows sample stable (RSD is 0.51%) in 2 hours, conclusion: at room temperature short-term placement of sample was stablized in 2 hours, and prompting should complete the mensuration of sample in 2 hours.
3.2.6 accuracy test
It is appropriate that precision takes deydrokaividing reference substance, and being made into concentration is the reference substance stock solution of 7.062mg/ml.Precision takes the corydalis saxicola bunting total alkali powder (lot number: 071014) 12.5mg of known content (56.47%) respectively, totally 9 parts, put in 50ml measuring bottle, add respectively above-mentioned reference substance stock solution 0.8ml, 1.0ml, 1.2ml, with 1% methanol hydrochloride solution, dissolve constant volume, accurate draw solution 1.0ml puts in 50ml measuring bottle, with 1% methanol hydrochloride solution, is settled to scale.Measure content.The results are shown in Table 9.
Table 9 accuracy test result
Result shows that average recovery rate is that 98.06%, RSD is 1.26%.Conclusion: the average recovery test findings of sample meets content assaying method and learns the requirement of investigating.
3.2.7 the establishment of corydalis saxicola bunting total alkali assay method condition
Through to methodological studies such as the range of linearity, precision, need testing solution stability, repeatability, the recovery, prove that this method is easy and simple to handle, accurate, feasible.With reference to ultraviolet spectrophotometry (appendix VA of < < Chinese Pharmacopoeia > > version in 2010), measure, now determine that the method for Determination of Total Alkaloid is as follows:
The learn from else's experience about 10mg of deydrokaividing reference substance of phosphorus pentoxide dried overnight of the preparation of reference substance solution, accurately weighed, put in 100ml measuring bottle, add 1% methanol hydrochloride solution to scale, shake up, as stock solution, standby.
The preparation precision of typical curve measures reference substance stock solution 0.3ml, 0.4ml, and 0.5ml, 0.6ml, 0.8ml, 1.0ml, puts respectively in 10ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up; Take 1% methanol hydrochloride solution as blank, measure absorbance log at 347nm wavelength place, take absorbance log as ordinate, concentration is horizontal ordinate, drawing standard curve.
The preparation precision of need testing solution takes this product 25mg in 50ml volumetric flask, adds 1% methanol hydrochloride solution to dissolve and constant volume, as sample stock solution, standby; The accurate sample stock solution 1.0ml that draws puts in 50ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up, and obtains.
Determination method is got need testing solution, take 1% methanol hydrochloride solution as blank, measures absorbance log in accordance with the law, from typical curve, reads the content of corydalis saxicola total alkaloid in need testing solution, calculates, and obtains.
3.2.8 three batch sample Determination of Total Alkaloid
By above-mentioned corydalis saxicola total alkaloid content assaying method, measure three batch sample content.The results are shown in Table 10.
Table 10 3 batch sample corydalis saxicola total alkaloid assay results
This product is respectively 56.82%, 56.74%, 56.24% containing corydalis saxicola total alkaloid.According to above result, tentative this product must not be lower than 50% containing corydalis saxicola total alkaloid content.
3.3 Scoulerines, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride, berberine hydrochloride content
3.3.1 chromatographic condition and system suitability test
Hanbon ODS-2C18 (250mm * 4.6mm, 5 μ m) is chromatographic column, and the acetonitrile-0.01mol/L potassium dihydrogen phosphate aqueous solution (18.5: 81.5) of take is mobile phase, 30 ℃ of column temperatures, detect wavelength 347nm, flow velocity 1.0ml/min, sample size 10 μ l, sampling time 50min.
3.3.2 the preparation of reference substance solution
It is appropriate that precision takes Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride, Berberine hydrochloride, adds methyl alcohol and make respectively single standard items storing solution.It is appropriate that precision is got each storing solution respectively, is mixed with hybrid standard product storing solution, and the mass concentration of above 6 compounds in hybrid standard product storing solution is respectively 0.0238,0.0889,0.0134,0.098,0.06292,0.0174mg/mL.
3.3.3 need testing solution preparation
Get the about 25mg of this product, accurately weighed, put in 50ml measuring bottle, add 1% hydrochloric acid methanol to scale, shake up, cross miillpore filter (0.45 μ m), get subsequent filtrate, obtain.
3.3.4 the range of linearity is investigated
The above-mentioned mixing reference substance solution 4 μ l of accurate absorption, 6 μ l, 8 μ l, 10 μ l, 14 μ l, 16 μ l sample introductions are analyzed, and each volume sample introduction 2 times, measures its peak area, averages.The sample size (X) of reference substance of take is horizontal ordinate, and peak area (Y) is ordinate, draws the typical curve of each reference substance, calculates the equation of linear regression of each composition.Result shows that each compound is good in respective range internal linear relation, in Table 11.
Table 11 linear test result
3.3.5 precision test
The same mixing reference substance solution of accurate absorption, continuous sample introduction 6 times, records each compound peaks area.Result shows, the RSD% of each compound peaks area is respectively 2.55,1.60,2.76,0.85,0.81,0.96, shows that instrument precision is good.The results are shown in Table 12.
Table 12 Precision test result
3.3.6 stability test
Respectively at 0,1,2,4,8,10 after preparation, 12,24h, the same need testing solution 10ul sample introduction of accurate absorption is measured peak area, and the RSD value of each compound stability is respectively 3.45%, 1.65%, 2.94%, 0.96%, 0.82%, 0.99%, at room temperature 24h is interior stable to show sample.The results are shown in Table 13.
Table 13 stability test result
3.3.7 replica test
Get with a collection of corydalis saxicola bunting total alkali 25mg, totally 6 parts, accurately weighed, by 3.4 below legal systems, for sample, the average content of measuring compound 1-7 is respectively 2.93%, 9.85%, 1.68%, 11.00%, 10.84%, 2.30%, RSD difference 1.57%, 2.63%, 3.42%, 2.05%, 1.98%, 2.41%, show that the method repeatability is good.
3.3.8 accuracy testing
Get the about 12.5mg of sample powder with a collection of known content, accurately weighed, totally 9 parts, put in 50ml measuring bottle, precision adds the reference substance of the content 80%, 100%, 120% that is equal to each reference substance in sample respectively, by 1.4 below legal system available test sample solutions, measure and calculate average recovery and the RSD of each composition.The results are shown in Table 14.
Table 14 average recovery test findings (n=3)
3.3.9 three batch sample assays
By above-mentioned content assaying method, measure three batch sample content.The results are shown in Table 15.
Table 15 external standard method assay result (n=3)
This product Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride, Berberine hydrochloride total content are respectively 34.07%, 38.04%, 37.76%.According to above result, the index component content summation in tentative this product corydalis saxicola total alkaloid must not be lower than 30.0%.
Claims (5)
1. a quality determining method for corydalis saxicola bunting total alkali, is characterized in that the method comprises the following steps:
(1) take meadowrueleaf corydalis root as control medicinal material, take deydrokaividing, palmatin hydrochloride, Berberine hydrochloride is reference substance, and corydalis saxicola bunting total alkali is carried out to thin layer discriminating;
(2) moisture of corydalis saxicola bunting total alkali, residue on ignition, heavy metal and arsenic salt are checked;
(3) adopt the content of total alkaloids in determined by ultraviolet spectrophotometry corydalis saxicola bunting total alkali;
(4) adopt the content of Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride and Berberine hydrochloride in hplc simultaneous determination corydalis saxicola bunting total alkali.
2. the quality determining method of corydalis saxicola bunting total alkali according to claim 1, is characterized in that the thin-layer identification method step of corydalis saxicola bunting total alkali is:
A. the preparation of need testing solution: get this product 10mg, add 25ml methyl alcohol to dissolve, ultrasonic processing 30 minutes, filters, and filtrate is as need testing solution;
B. the preparation of control medicinal material solution: get meadowrueleaf corydalis root control medicinal material 0.1g, add 5ml methyl alcohol, ultrasonic processing 30 minutes, filters, and filtrate is medicinal material solution in contrast;
C. the preparation of reference substance solution: get deydrokaividing, palmatin hydrochloride, Berberine hydrochloride reference substance, add methyl alcohol and make every 1ml containing the mixed liquor of deydrokaividing 0.2mg, palmatin hydrochloride 0.2mg, Berberine hydrochloride 0.1mg, as mixing reference substance solution;
D. thin layer condition and result: the thin-layered chromatography of recording according to appendix VI B of Chinese Pharmacopoeia version in 2010 is tested, draw each 5 μ l of above-mentioned three kinds of solution, point be take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder in same, take ratio as 9: 2: 5: ethyl acetate-methylene chloride-methanol-strong ammonia solution of 3, and normal butyl alcohol-glacial acetic acid-water that ratio is 7: 1: 2 is developping agent, launch, take out, dry up, putting wavelength is to inspect under 365nm ultraviolet lamp, in test sample chromatogram, with reference substance and the corresponding position of control medicinal material chromatogram on, the fluorescence spot of aobvious same color.
3. the quality determining method of corydalis saxicola bunting total alkali according to claim 1, is characterized in that the inspection method step of corydalis saxicola bunting total alkali moisture, residue on ignition, heavy metal and arsenic salt is:
A. the aquametry of recording according to appendix IX H first method of Chinese Pharmacopoeia version in 2010 is measured the moisture of corydalis saxicola bunting total alkali, and in corydalis saxicola bunting total alkali, moisture must not surpass 5.0%;
B. the residue on ignition determination method of recording according to appendix IX J of Chinese Pharmacopoeia version in 2010 is measured the residue on ignition of corydalis saxicola bunting total alkali, and in corydalis saxicola bunting total alkali, residue on ignition must not cross 2.0%;
C. the heavy metal inspection method that appendix IX E of Chinese Pharmacopoeia version in 2010 the second method is recorded is measured, the ancient Cai Shi method of first method that the inspection of arsenic salt is recorded by appendix IX F of Chinese Pharmacopoeia version in 2010 is measured, in corydalis saxicola bunting total alkali, heavy metal must not cross 10/1000000ths, and arsenic salt must not cross 2/1000000ths.
4. the quality determining method of corydalis saxicola bunting total alkali according to claim 1, is characterized in that the content step of total alkaloids in determined by ultraviolet spectrophotometry corydalis saxicola bunting total alkali is:
A. the preparation of the reference substance solution about 10mg of deydrokaividing reference substance of phosphorus pentoxide dried overnight that learns from else's experience, accurately weighed, puts in 100ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up, as stock solution, standby;
B. the preparation precision of typical curve measures reference substance stock solution 0.3ml, 0.4m1, and 0.5ml, 0.6ml, 0.8ml, 1.0ml, puts respectively in 10ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up; Take 1% methanol hydrochloride solution as blank, and the UV-VIS spectrophotometry of recording according to appendix V A of Chinese Pharmacopoeia version in 2010, measures absorbance log at 347nm wavelength place, take absorbance log as ordinate, concentration be horizontal ordinate drawing standard curve;
C. the about 25mg of this product is got in the preparation of need testing solution, accurately weighed, puts in 50ml volumetric flask, adds 1% methanol hydrochloride solution to scale, as sample stock solution, standby; The accurate sample stock solution 1.0ml that draws puts in 50ml measuring bottle, adds 1% methanol hydrochloride solution to scale, shakes up, and obtains;
D. determination method: get need testing solution, take 1% methanol hydrochloride solution as blank, measure absorbance log in accordance with the law, the amount of reading deydrokaividing in need testing solution from typical curve, calculates, and obtains.This product in deydrokaividing C21H23O4N, must not be less than 50.0% containing total alkaloids.
5. the quality determining method of corydalis saxicola bunting total alkali according to claim 1, is characterized in that the content step of Scoulerine in hplc simultaneous determination corydalis saxicola bunting total alkali, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride and Berberine hydrochloride is:
A. chromatographic condition and system suitability adopt Hanbon ODS-2C18, specification is 250mm * 4.6mm, the chromatographic column of 5 μ m, acetonitrile-0.01mol/L potassium dihydrogen phosphate aqueous solution that the ratio of take is 18.5: 81.5 is mobile phase, 30 ℃ of column temperatures, detect wavelength 347nm, flow velocity 1.0ml/min, sample size 10 μ l, sampling time 50min;
B. it is appropriate that the preparation precision of reference substance solution takes Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, deydrokaividing, palmatin hydrochloride, Berberine hydrochloride, add methyl alcohol and make respectively single standard items storing solution, it is appropriate that precision is got each storing solution respectively, be mixed with hybrid standard product storing solution, the mass concentration of above 6 compounds in hybrid standard product storing solution is respectively 0.0238,0.0889,0.0134,0.098,0.06292,0.0174mg/mL;
C. the about 25mg of this product is got in the need testing solution preparation of preparing of need testing solution, accurately weighed, puts in 50ml measuring bottle, adds 1% hydrochloric acid methanol to scale, shakes up, and crosses 0.45 μ m diameter miillpore filter, gets subsequent filtrate, obtains;
D. determination method: accurate respectively 10 μ l of reference substance and need testing solution that draw respectively, injection liquid chromatography, measure, obtain, this product must not be less than 32.0% containing Scoulerine, dehydrogenation first Ka Weiting, jateorrhizine, dehydrocavidine, palmatin hydrochloride, Berberine hydrochloride total amount.
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