CN1973854A - Process of preparing motherwort injection - Google Patents
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Abstract
The present invention relates to motherwort injection and its high quality preparation process. The preparation process includes optimized extracting step, and has raised injection consistency and high and stable product quality.
Description
Technical field
The present invention relates to high-quality motherwort injection and preparation method thereof.
Background technology
Chinese crude drug is adopted the influence of factors such as date because of seed, the place of production and growth conditions, receipts, and its quality often exists than big-difference, and the also corresponding existence of the composition of inherent material and ratio is than big-difference; Usually in the tcm product, its active substance basis not only is subjected to the influence of medical material, and the reasonability of technological parameter and fluctuation also have a strong impact on the concordance of inherent component and proportion of composing.There is very big difference in the Chinese medicine in part tcm product, the particularly Chinese medicine different production of same kind period because of these uncontrollable factors make.In recent years; because people all increase substantially the level of understanding and the level of consumption of medicine; negative evaluation to Chinese medicine injection is in rising trend; Chinese medicine injection has been absorbed among the curative effect trust crisis; standard improves the level of production of Chinese medicine; the control level that improves the quality improves the quality standard of Chinese medicine, and the cry of standard Chinese medicine is surging.Bureau of Drugs Supervision of country carries out the research of injection finger printing in calendar year 2001 decision, to improve the quality level of Chinese medicine.This shows, produce high-quality tcm product for example Chinese medicine be desired always.
At present, domestic existing people has proposed some and has adopted Chinese medicine fingerprint to control the imagination of tcm product quality, and for example, Sha Ming etc. mention HPLC dactylogram technology is applied to the new Chinese medicine quality control, Chinese herbal medicine, 33 (2), 8-10 page or leaf, 2002; Xie Peishan has proposed the research and the application of chromatographic fingerprints of Chinese materia medica quality control pattern, the modernization of Chinese medicine of World Science technology, 3 (6), 28-31 page or leaf, 2001; Tu Pengfei mentions the enforcement and the Chinese crude drug GAP construction of base of Chinese medicine injection finger printing examination criteria, Chinese medicine research and information, 3 (10), 17-18 page or leaf.But, still there is quite difficult road to walk because the multiformity of herbal species is specialized fingerprint pattern technology, and is applied on the suitability for industrialized production of a certain concrete Chinese crude drug product.
Motherwort injection has had some correlational study reports.Zhang Chongyi has reported the preparation and the Study on Quality Control of motherwort injection, wherein relates to the content of Herba Leonuri injection quality control standard, the 1984 (9), the 12nd page of Chinese patent medicine research; CN1456548A, CN1415603A relate to a kind of leonurine salt and preparation thereof, wherein disclose employing Herba Leonuri total alkali or its alkali salt prepares the Herba Leonuri injection; CN1530126A, CN1262118A relate to a kind of pharmaceutical composition that contains Herba Leonuri, and said composition can be made into injection or other preparation, is used for the treatment of gynaecopathia and chronic renal failure.
About the assay method of stachydrine hydrochloride, the pertinent literature report is arranged equally also.Chinese Pharmacopoeia (one one) recorded the quantitative approach (is contrast with the stachydrine hydrochloride) of Reinecke salt residue colorimetric method for determining total alkaloids amount in 2000; Jiang Shunyao has reported stachydrine composition in employing high performance liquid chromatography (HPLC) the analysis Herba Leonuri medical material, pharmaceutical analysis magazine, the 21 (4), the 243rd page, 2001; Song Jingping etc. have also done research and report, Guangxi medicine, the 24 (8), the 1289th page, 2002 to the HPLC analytic process of the stachydrine hydrochloride of Herba Leonuri.
The same with other tcm product, how to improve the quality of motherwort injection, be that people expect the problem that solves always.The present inventor is by carrying out deeply thin young research to motherwort injection, established motherwort injection HPLC finger printing standard, global feature material base by the locking motherwort injection, optimize extraction process, improve the concordance of injection global feature material base, make the constant product quality unanimity, thereby produce high-quality motherwort injection, so finished the present invention.
Summary of the invention
One of purpose of the present invention provides a kind of high-quality motherwort injection.
Another object of the present invention provides a kind of preparation method of producing high-quality motherwort injection.
A further object of the present invention provides the assay method of the stachydrine hydrochloride in the Herba Leonuri, is used to control the product quality of motherwort injection.
The Herba Leonuri injection is commercially available tcm product, and existing product was issued two operative norms, the one, and Chinese Pharmacopoeia " 1977 editions " standard, the motherwort injection of the 493rd page of promulgation:
The sterile water solution that this product is made for the labiate Herba Leonuri.Contain total alkaloids with stachydrine hydrochloride (C
7H
13NO
2HCl) calculate, should be the 90-110% of labelled amount.
This product can add the benzoic acid of 0.5-1.0%.
[inspection] pH value should be 4.5-6.5 (42 pages of appendix).
[assay] measures trap according to colorimetry (32 pages of appendix) at the wavelength place of 525nm, is ordinate with the trap, and concentration is abscissa, the drawing standard curve.From standard curve, read the content in the need testing solution, calculate content.
[specification] 1ml:20mg.
Convert by its acceptability limit that can fluctuate, every of this product contains total alkaloids with stachydrine hydrochloride (C
7H
13NO
2HCl) calculate, should be 18-22mg.
Another is present operative norm, the Herba Leonuri injection of issuing among the 20 WS3-B-3969-98 of Ministry of Public Health Chinese traditional patent formulation preparation:
This product is the sterile water solution of Herba Leonuri through being processed into.
[method for making] got Herba Leonuri and decocted with water three times, and collecting decoction filters, it is 1.36～1.38 (80 ℃) that filtrate decompression is concentrated into relative density, and thin up to water content is about 35%, respectively with the ethanol extraction of 88%-95% three times, leave standstill, filter, reclaim ethanol, it is colourless to solution to decolour with active carbon (consumption is 0.01%-0.5%), adds 1% benzyl alcohol, adds the injection water and makes into and contain stachydrine 20mg among the 1ml, take off charcoal, fine straining, embedding, sterilization, promptly.
[assay]
Total alkaloids: according to spectrophotography (appendix VB), the wavelength place of D525nm measures trap respectively, deducts the trap of reference substance and test sample respectively with the trap of blank reagent, calculating, promptly.
[specification] 1ml:20mg.
Convert by its acceptability limit that can fluctuate, every of this product contains total alkaloids with stachydrine hydrochloride (C
7H
13NO
2HCl) calculate, should be 18-22mg.
The Chinese medicine of approved listing is because the quality of injection Chinese crude drug is subjected to the influence of factors such as seed source, the place of production, collection period; Owing to be subjected to the influence of production technology fluctuation, the quality stability of traditional medicine Injectio did not well guarantee in the period of growth, and the every controlling index and the means of existing quality standard can not reach effective control to the quality of injection.
Equally, for motherwort injection, though provided in the above-mentioned existing standard and contained total alkaloids should be 18-22mg with stachydrine hydrochloride calculating content standard in every 1ml injection, consider the Chinese medicine preparation complicated component, the influence factor is more, select the colorimetrically analysing of stipulating in the existing standard for use, under the satisfactory situation of measuring of stachydrine hydrochloride content, can not guarantee the concordance of injection quality.
The present inventor is according to " requirement of Chinese medicine fingerprint pattern technology " of national Bureau of Drugs Supervision promulgation and the thinking of " Chinese medicine finger printing research guide ", adopt the HPLC method, research has also been established Herba Leonuri injection finger printing, in order to the quality of control product.Among the present invention, adopting good, the highly sensitive HPLC method of selectivity, present operative norm is replenished, guaranteeing the quality of motherwort injection, is of great advantage to the raising of motherwort injection quality.
The present invention adopts the HPLC method, is isolation medium mutually with the ion-type bonding, by including ion exchange at interior mixing retention mechanism, makes that stachydrine hydrochloride is separated in the motherwort injection, thereby measures its content.
Moreover, the present invention improves the quality of motherwort injection also by the technology of composition change and optimization.
In specific embodiment, motherwort injection of the present invention is the sterile water solution of Herba Leonuri through being processed into.The preparation technology of product of the present invention is as follows: get Herba Leonuri and decoct with water repeatedly, for example 2-5 time, collecting decoction filters, it is 1.36～1.38 (80 ℃) that filtrate decompression is concentrated into relative density, extracts with Different concentrations of alcohol respectively, leaves standstill, filter, merging filtrate, it is colourless or faint yellow to solution to decolour with proper amount of active carbon, leave standstill, filter, reclaim ethanol, add the injection water and make, for example become to contain among the 1ml the about 20mg of total alkaloids, fine straining to the desired concn specification, embedding, sterilization, promptly.Unlike the prior art be, technology of the present invention is adopting aqueous extraction-alcohol precipitation technology to make in the medicinal liquid, promptly in the filtrate that He Bings, add for example (consumption is the 0.01%-0.5w/v% of amount of liquid medicine) depyrogenation of proper amount of active carbon, decolouring, add adsorbent simultaneously, adsorbent is for example to be selected from Talcum, bentonite, in the mineral such as kieselguhr and potter's clay one or more, preferably contain the activatory Talcum of having sterilized: kieselguhr (1: 0.1-10), by 0.5%-5% (in amount of liquid medicine, w/v%) be mixed in the medicinal liquid and deposit a period of time, can be for example 12-48 hour, get final product in 12-24 hour usually, optionally, this section period can be lacked slightly or is longer, filter, reclaim ethanol, add the injection water then and make to the desired concn specification, fine straining, embedding, sterilization, promptly.Do not add benzyl alcohol in the product preparation.Described activation is meant: the processing of high temperature or other physical conditions.
Existing process using ethanol remove impurity, specificity is poor, and in remove impurity, its active component content declines to a great extent, and the ion that need remove then needs repeatedly precipitate with ethanol also to be difficult for removing.Technology of the present invention then combines alcohol precipitation process with single-minded absorbing process, strict control potassium ion significantly improves product quality.Though active carbon is also had an absorption property, the adsorbent that provides of the present invention can significantly improve the various performance indications of product, thereby promotes the quality of product.
Specifically, product of the present invention has been removed potassium ion in the injection, and has been cancelled the benzyl alcohol that former Herba Leonuri injection adds because of intramuscular injection pain, and had the effect that alleviates injection pain owing to process optimization.
Product of the present invention is a kind of high-quality injection, and this injection physicochemical property is stable, and its clarity is high and microgranule is few, contains the above microgranule of 10 μ m≤4000 in every test sample, contains the above microgranule of 25 μ m≤400.The particulate matter detection method is: sample thief 50ml, and according to " 2005 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, the light blockage method inspection.Compare in every test sample of existing product and contain the above microgranule of 10 μ m less than 6000, contain the above microgranule of 25 μ m less than 600, quality is greatly improved.
In addition, product of the present invention also has following performance:
Protein is got this product 1ml, adds 30% sulfosalicylic acid test solution 1ml of new preparation, mixes.Placed 5 minutes, and do not occur muddy, up to specification;
Tannin is got this product 1ml, adds the physiological sodium chloride solution 5ml that contains 1% Ovum Gallus domesticus album of new preparation, places 10 minutes, does not occur muddy or precipitation, and is up to specification;
Resin is got this product 5ml, adds 1 of hydrochloric acid, places 30 minutes, precipitation do not occur, and is up to specification;
Oxalates: it is an amount of to get this product, regulates pH value to 1～2 with dilute hydrochloric acid, filters, and gets filtrate 2ml, and filtrate is regulated PH to 5～6, adds 2～3 of 3% calcium chloride solutions.Placed 10 minutes, and do not occur muddy or precipitation.
Determining with the assay condition of finger printing of the present invention is as follows:
Finger printing is according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000 measures) and " Chinese medicine chromatographic fingerprinting experimentation technical manual (trying) "
Chromatographic condition and system suitability test apparatus: Waters 2690 chromatograph of liquid; Chromatographic column: Aichrom Bond-AQ C18 4.6mm * 250mm, 5 μ m; Mobile phase: 0.1mol/L sodium dihydrogen phosphate (transferring pH to 5.5) with 0.1% phosphoric acid; Column temperature: 30 ℃; Wavelength: 242nm; Test with retinue system object of reference, the chromatographic peak of appearance should be no less than with reference to collection of illustrative plates, and figure should be similar.Continuous sample introduction 5 pins, the similarity between its figure should be not less than 0.99.
One of retinue object of reference is got in the preparation of retinue system object of reference solution, adds 300 μ l water and makes dissolving, promptly.The standard specimen of the synthetic standard fingerprint image of retinue object of reference.
The preparation of need testing solution: motherwort injection is as need testing solution.
Accurate respectively retinue system's object of reference solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram is provided with integral parameter, the integration peak area; With Chinese Pharmacopoeia Commission's chromatographic fingerprints of Chinese materia medica similarity computed in software, the similarity of motherwort injection standard fingerprint image and this product should be not less than 0.9.
The finger printing of determining is a feedstock capture in the motherwort injection production process, and intermediate, finished product production and product stability quality control standard collection of illustrative plates have specificity and originality.Detect with no benzyl alcohol peak existence in the product of prepared of the present invention with the standard fingerprint image.
[assay] total alkaloids
The preparation precision of reference substance solution takes by weighing the stachydrine hydrochloride reference substance 25mg that is dried to constant weight at 105 ℃, puts in the 25ml measuring bottle, with the 0.1mol/l dissolve with hydrochloric acid solution and be diluted to scale, shakes up, and promptly gets (the hydrochloric stachydrine 1mg of every 1ml).
The preparation precision of need testing solution is measured this product 5ml, puts in the 100ml measuring bottle, with 0.1mol/L hydrochloric acid solution degree of being diluted to, shakes up, promptly.
Algoscopy precision is respectively measured reference substance solution, need testing solution, each 10ml of 0.1ml/L hydrochloric acid solution, place three 25ml measuring bottles respectively, each accurate 2% chromic thiocyanate ammonium salt solution 3ml that adds new system shakes up, and adds the 0.1mol/L hydrochloric acid solution to scale, shake up, put in the ice bath and placed 1 hour, filter, discard filtrate just, getting subsequent filtrate, is blank with the 0.1mol/L hydrochloric acid solution.According to spectrophotography (appendix VB), the wavelength place of D525nm measures trap respectively, deducts the trap of reference substance and test sample respectively with the trap of blank reagent, calculates, promptly.
Stachydrine hydrochloride is according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005 measures)
Chromatographic condition and system suitability test are filler with the cationite of sulfonic acid group bonded silica gel (SCX); With 0.05 ± 0.02mol/L potassium dihydrogen phosphate-triethylamine 100: 0.1 ± 0.05, be mobile phase with phosphoric acid adjust pH to 2.3 ± 0.5; The detection wavelength is 203nm.Number of theoretical plate calculates by the stachydrine hydrochloride peak should be not less than 1500.
It is an amount of that the preparation precision of reference substance solution takes by weighing the stachydrine hydrochloride reference substance, adds mobile phase and be mixed with the solution that every 1ml contains 0.4mg, promptly.
The preparation precision of need testing solution is measured this product 1ml, puts in the 25ml measuring bottle, is diluted to scale with mobile phase, shakes up, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
Product of the present invention has accompanying drawing 2 described standard diagrams.And when spectrophotography is surveyed and contained total alkaloids in every 1ml injection and be 20mg with the stachydrine hydrochloride, adopt the HPLC method to measure hydrochloric stachydrine and be no less than 8mg.
Description of drawings
Fig. 1 is a stachydrine hydrochloride reference substance collection of illustrative plates.
Fig. 2 is a Herba Leonuri injection standard finger-print.
Fig. 3 is a Herba Leonuri injection standard curve.
Fig. 4 is the liquid chromatogram of Herba Leonuri injection of the present invention.
Fig. 5 is the liquid chromatogram of 3 kinds of commercially available Herba Leonuri injection.
The specific embodiment
The preparation of embodiment 1 Herba Leonuri injection
Getting Herba Leonuri decocts with water three times, collecting decoction, filter, it is 1.36-1.38 (80 ℃) that filtrate decompression is concentrated into relative density, thin up to water content is about 35%, with the ethanol extraction of 88%-95% concentration three times, leaves standstill respectively, filter, reclaim ethanol, add the active carbon (consumption is 0.1%) and the activatory Talcum of having sterilized: it is colourless or faint yellow to solution to decolour in the adsorbent of kieselguhr (1: 1) (consumption is 1%) the mixture medicinal liquid, and after depositing 24 hours, adding the injection water makes into and contains the about 20mg of total alkaloids among the 1ml, take off charcoal and adsorbent, fine straining, embedding, sterilization promptly gets the Herba Leonuri injection.According to spectrophotography (appendix VB), D525nm wavelength place measures, and biology total alkali is 20mg/ml in stachydrine hydrochloride content, adopts the HPLC method that provides among the embodiment 3 to measure, and stachydrine hydrochloride content is 13.78mg/ml.Adopt the finger printing chromatographic condition of embodiment 4 to measure, the collection of illustrative plates of measuring Chinese Pharmacopoeia Commission's chromatographic fingerprints of Chinese materia medica similarity computed in software, the similarity of this product and standard finger-print is 0.99.
This injection physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter sample thief 50ml, according to " 2005 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, the light blockage method inspection contains the above microgranule of 10 μ m less than 4000 in every test sample, contain the above microgranule of 25 μ m less than 400.
Protein is got this product 1ml, adds 30% sulfosalicylic acid test solution 1ml of new preparation, mixes.Placed 5 minutes, and do not occur muddy, up to specification;
Tannin is got this product 1ml, adds the physiological sodium chloride solution 5ml that contains 1% Ovum Gallus domesticus album of new preparation, places 10 minutes, does not occur muddy or precipitation, and is up to specification;
Resin is got this product 5ml, adds 1 of hydrochloric acid, places 30 minutes, precipitation do not occur, and is up to specification;
Oxalates: it is an amount of to get this product, regulates pH value to 1～2 with dilute hydrochloric acid, filters, and gets filtrate 2ml, and filtrate is regulated PH to 5～6, adds 2～3 of 3% calcium chloride solutions.Placed 10 minutes, and do not occur muddy or precipitation.
The preparation of embodiment 2 Herba Leonuri injection
Getting Herba Leonuri decocts with water three times, collecting decoction, filter, it is 1.36-1.38 (80 ℃) that filtrate decompression is concentrated into relative density, thin up to water content is about 35%, with the ethanol extraction of 88%-95% concentration three times, leaves standstill respectively, filter, reclaim ethanol, add active carbon (consumption is 0.5%) and contain the activatory Talcum of sterilizing: it is colourless or faint yellow to solution to decolour in the adsorbent of kieselguhr (1: 1) (consumption is 3%) the mixture medicinal liquid, and after depositing 24 hours, adding the injection water makes into and contains the about 20mg of total alkaloids among the 1ml, take off charcoal and adsorbent, fine straining, embedding, sterilization promptly gets the Herba Leonuri injection.According to spectrophotography (appendix VB), D525nm wavelength place measures, and biology total alkali is 20mg/ml in stachydrine hydrochloride content, adopts the HPLC method that provides among the embodiment 3 to measure, and stachydrine hydrochloride content is 13.46mg/ml.Adopt the finger printing chromatographic condition of embodiment 4 to measure, the collection of illustrative plates of measuring Chinese Pharmacopoeia Commission's chromatographic fingerprints of Chinese materia medica similarity computed in software, the similarity of this product and standard finger-print is 0.98.
This injection physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter sample thief 50ml, according to " 2005 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, the light blockage method inspection contains the above microgranule of 10 μ m less than 4000 in every test sample, contain the above microgranule of 25 μ m less than 400.
Protein is got this product 1ml, adds 30% sulfosalicylic acid test solution 1ml of new preparation, mixes.Placed 5 minutes, and do not occur muddy, up to specification;
Tannin is got this product 1ml, adds the physiological sodium chloride solution 5ml that contains 1% Ovum Gallus domesticus album of new preparation, places 10 minutes, does not occur muddy or precipitation, and is up to specification;
Resin is got this product 5ml, adds 1 of hydrochloric acid, places 30 minutes, precipitation do not occur, and is up to specification;
Oxalates: it is an amount of to get this product, regulates pH value to 1～2 with dilute hydrochloric acid, filters, and gets filtrate 2ml, and filtrate is regulated PH to 5～6, adds 2～3 of 3% calcium chloride solutions.Placed 10 minutes, and do not occur muddy or precipitation.
The HPLC of embodiment 3 Herba Leonuri injection stachydrine hydrochlorides detects
(1) experimental apparatus and reagent
(1) instrument:
The Waters717-Waters2487-M32 work station
The Waters2690-Waters2487-M32 work station
Chromatographic column: Alltech SCX 5 μ (4.6 * 250mm)
Phenosphere SCX 5μ(4.6×250mm)
MILLIPORE ultra-pure water preparing instrument
(2) reagent
Sample is provided by No.1 Shidai Medicine Inst. Co., Ltd., Chengdu.
Reference substance China pharmaceutical biological product check institute (stachydrine hydrochloride, lot number: 712-9903 use for assay)
The phosphorylating reagent potassium dihydrogen is that Chengdu chemical reagent factory produces, and water is ultra-pure water, and all the other reagent are analytical pure.
(2) chromatographic condition
(1) detects wavelength: 203nm.
(2) analytical column Alltech SCX 5 μ (4.6 * 250mm) cation-exchange chromatography posts, with 0.05mol/L potassium dihydrogen phosphate-triethylamine (100: 0.1) (transferring pH to 2.3 with phosphoric acid) is mobile phase, the result is at 203nm wavelength place, reference substance and sample all have maximum absorption band, stachydrine hydrochloride peak and adjacent peak separating degree are greater than 1.5 in the sample, and theoretical cam curve is calculated all greater than 2000 with the stachydrine hydrochloride peak.
(3) mobile phase is mobile phase with 0.05mol/L potassium dihydrogen phosphate-triethylamine (100: 0.1) (transferring pH to 2.3 with phosphoric acid), flow velocity 1ml/min, column temperature: 30 ℃.Reference substance, test sample chromatogram are seen accompanying drawing 1,2.
(4) evaluation of chromatographic condition
By different people, chromatographic system and object of study chromatographic condition and system suitability are investigated, the result is as follows:
1. separating degree: the peak energy of stachydrine hydrochloride reaches good separating with other impurity peaks in the sample, separating degree R 〉=1.5.
2. number of theoretical plate: the number of theoretical plate at stachydrine hydrochloride peak is not less than 2000 in reference substance and the sample.
(5) conclusion is a filler with the cationite of sulfonic acid group bonded silica gel (SCX); With 0.05mol/L potassium dihydrogen phosphate-triethylamine (100: 0.1) (with phosphoric acid adjust pH to 2.3) is mobile phase; The detection wavelength is 203nm.Number of theoretical plate is not less than 2000 chromatographic condition by the stachydrine hydrochloride peak can be satisfied and detect requirement.
(3) preparation of need testing solution
Because sample is an injection, strict control is all arranged on clarity and microgranule, and keep the physical presence state of each material in the injection as far as possible, so we adopt sample directly to dilute the method for back sample introduction.
(4) stability test
Sample stability: by same sample is placed 0,1,2,4,8,24 hour investigation in room temperature, its stability better, RSD=1.4% shows that need testing solution measured in 24 hours, the result stablizes.The results are shown in Table 1.
The investigation of table 1 Herba Leonuri injection stability
| Standing time (h) | 0 | 1 | 2 | 4 | 8 | 24 |
| The sample peak area | 3851403 | 3839708 | 3788879 | 3731918 | 3719489 | 3763911 |
| Average peak area | 3782551 | |||||
| RSD(％) | 1.4％ | |||||
Reference substance solution stability: by with concentration be the investigation that the stachydrine hydrochloride reference substance solution of 0.0816mg/ml was placed 0,1,2,4,8,24 hour under the condition of room temperature, its stability better, RSD=1.25% shows that reference substance solution measured in 24 hours, the result is stable.The results are shown in Table 2.
The investigation of table 2 reference substance solution stability
| The stability of stachydrine hydrochloride reference substance solution | ||||||
| Standing time (h) | 0 | 1 | 2 | 4 | 8 | 24 |
| Peak area | 275629 | 283671 | 279973 | 285558 | 283399 | 281888 |
| Average peak area | 281686 | |||||
| RSD(％) | 1.25％ | |||||
(5) methodological study
(1) reference substance solution precision
The stachydrine hydrochloride reference substance solution 10 μ l that accurate absorption concentration is 0.452mg/ml inject chromatograph of liquid 5 times, and the record chromatogram by calculated by peak area, the results are shown in Table 3.Illustrate that sample introduction precision can satisfy the assay requirement of this product.
The precision of table 3 stachydrine hydrochloride reference substance solution
| Sequence number |
| 1 | 2 | 3 | 4 | 5 | |
| Peak area | 1532074 | 1587763 | 1531513 | 1578060 | 1586092 |
| Average peak area | 1563100 | ||||
| RSD(％) | 1.84％ |
(2) range of linearity
Accurate absorption concentration is stachydrine hydrochloride reference substance solution 5 μ l, 10 μ l, 20 μ l, 30 μ l, the 40 μ l of 0.328mg/ml, injects chromatograph of liquid respectively, the record chromatogram.The results are shown in Table 4, is abscissa with the peak area, and stachydrine hydrochloride reference substance sample size is a vertical coordinate, and the drawing standard curve is seen figure, gets regression equation.Y＝2.8637E-06X+0.6571，r＝0.9996。Show that stachydrine hydrochloride has good linear relationship in 1.64～13.12mg scope.
Table 4 stachydrine hydrochloride reference substance linear test result
| Sample size (μ g) | 1.64 | 3.28 | 6.56 | 9.84 | 13.12 |
| Peak area | 392850 | 911108 | 2004478 | 3169416 | 4401322 |
| Standard curve | Y＝2.8637E-06X+0.6571 | ||||
| Correlation coefficient | 0.9996 | ||||
| The range of linearity | 1.64～16.4ug | ||||
(3) replica test
Get Herba Leonuri injection (lot number 040807) and press under the assay item test sample preparation method operation repetitive 5 times, and measure, the results are shown in table 5.
Table 5 replica test result
| The | 1 | 2 | 3 | 4 | 5 |
| Sample size (mg/ml) | 14.50 | 14.42 | 14.54 | 14.39 | 14.46 |
| Average content (mg/ml) | 14.46 | ||||
| RSD(％) | 0.41 | ||||
(4) recovery test
(lot number is 040806 to accurate absorption sample, stachydrine hydrochloride content is 13.04mg/ml) 0.5ml, totally 5 parts, the accurate respectively stachydrine hydrochloride reference substance 6.32mg that adds, thin up is to 1ml, press stachydrine hydrochloride content assaying method mensuration under the quality standard text assay item, the formula calculate recovery rate the results are shown in Table 6 calculated as described below.It is as shown in the table, and it is 98.0% that this method application of sample reclaims average recovery rate, and RSD is 1.8%, and the accuracy of this extracting method is better as can be known by result of calculation.
Table 6 average recovery result of the test
| N O | Peak area | Sample volume (ml) | Stachydrine hydrochloride content (mg) in the sample | Reference substance addition (mg) | Actual measurement hydrochloric acid Soviet Union alkali content (mg) | The response rate (%) | Average recovery rate (%) | RSD( ％) |
| 1 | 1749608 | 0.5 | 6.52 | 6.32 | 12.76 | 98.62 | 98.0 | 1.8 |
| 2 | 1738670 | 0.5 | 6.52 | 6.32 | 12.68 | 97.36 | ||
| 3 | 1726372 | 0.5 | 6.52 | 6.32 | 12.59 | 95.94 | ||
| 4 | 1768521 | 0.5 | 6.52 | 6.32 | 12.89 | 100.6 8 | ||
| 5 | 1739921 | 0.5 | 6.52 | 6.32 | 12.68 | 97.36 |
The assay of (5) ten six batch samples
By detection method of the present invention, measure 16 batch samples, it the results are shown in Table 7.
Table 7 16 batch sample assay results
| Lot number | Measurement result (mg/ml) | Meansigma methods (mg/ml) | |
| 1 | 2 | ||
| 040801 | 12.09 | 12.50 | 12.30 |
| 040802 | 10.08 | 10.05 | 10.07 |
| 040803 | 13.71 | 13.86 | 13.78 |
| 040804 | 14.69 | 14.36 | 14.52 |
| 040805 | 13.13 | 13.32 | 13.22 |
| 040806 | 13.02 | 13.06 | 13.04 |
| 040807 | 14.57 | 14.76 | 14.67 |
| 040901 | 13.70 | 13.94 | 13.82 |
| 040902 | 13.46 | 14.09 | 13.77 |
| 040903 | 13.60 | 13.31 | 13.46 |
| 050109-1 | 14.72 | 14.57 | 14.65 |
| 050109-2 | 10.11 | 10.36 | 10.23 |
| 050109-3 | 10.60 | 10.70 | 10.65 |
| 050109-4 | 10.62 | 10.58 | 10.60 |
| 050109-5 | 10.17 | 10.34 | 10.26 |
| 050109-6 | 13.31 | 13.13 | 13.22 |
According to 16 batch sample measurement results as seen, the assay limit of this product is that the hydrochloric stachydrine of every 1ml is greater than 8mg.
Determining of embodiment 4 finger printing
[finger printing] is according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000 measures) and " Chinese medicine chromatographic fingerprinting experimentation technical manual (trying) ".
(1) instrument, equipment
Use same root chromatogram column, under the identical situation of column temperature, mobile phase, detection wavelength, on the instrument of two kinds of models, test respectively.
1, Waters 2690 type high performance liquid chromatographs, Waters 2487 detectors;
Continuous sample introduction is five times on the same day, and the retention time repeatability of chromatographic peak is fine.
2, Agilent 1100 type high performance liquid chromatographs: continuous sample introduction on the same day five times, the retention time repeatability of chromatographic peak is fine.
Result of the test on the instrument of two kinds of models shows, the repeatability that the high performance liquid chromatograph of use Waters and Agilent is measured is all good, the mensuration that all can be used for Herba Leonuri injection water soluble ingredient finger printing, but the former peak shape is better, so preferred Waters 2690 type high performance liquid chromatographs.
(2) chromatographic condition and system suitability test apparatus: Waters 2690 chromatograph of liquid; Chromatographic column: Aichrom Bond-AQ C
184.6mm * 250mm, 5 μ m; Mobile phase: 0.1mol/L sodium dihydrogen phosphate (transferring pH to 5.5) with 0.1% phosphoric acid; Column temperature: 30 ℃; Wavelength: 242nm; Test with retinue system object of reference, the total chromatographic peak of appearance should be no less than with reference to collection of illustrative plates, and figure should be similar.Continuous sample introduction 5 pins, the similarity between its figure should be not less than 0.99.
One of retinue object of reference is got in the preparation of retinue system object of reference solution, adds 300 μ l water and makes dissolving, promptly.
The preparation of need testing solution is got the Herba Leonuri injection as need testing solution.
Accurate respectively retinue system's object of reference solution and each the 10 μ l of need testing solution of drawing of algoscopy, inject chromatograph of liquid, record chromatogram to 15 minute is provided with integral parameter, and the peak area that is about 0.4% chromatographic peak with the peak area percentage rate is made as the smallest peaks area value; Similarity with Chinese Pharmacopoeia Commission's chromatographic fingerprints of Chinese materia medica similarity computed in software this product and standard finger-print should be not less than 0.9.
(3) the expression content explanation of finger printing standard
The main contents of finger printing standard comprise the description of instrument model and system suitability, assay method, and similarity is calculated, 5 contents of similarity limit.Wherein system suitability is the key that the decision standard runs succeeded, and it can not be described by the method for assay, and its evaluation index comprises chromatographic condition, testing conditions, writing time, date processing parameter, the stability of system etc.; Assay method comprises need testing solution preparation, sample injection method, similarity calculating (key is the selection and the computational methods of software for calculation); The similarity limit decides according to the complexity of the component of the stability of finished product, the repeatability of assay method, determined sample.
1, chromatographic condition and system suitability
The mensuration of finger printing requires very high to the parameter control of chromatographic condition and instrument, the chromatography column that uses in this standard is the aqueous post, because the stability of aqueous post is not as good as octadecyl silane post commonly used, therefore in the system suitability, investigate chromatographic column with sample introduction precision and whether reach the balance requirement; Use instrument to be defined as 2690 or 2695 of WATERS company production, detector is 2487, measure for VWD G1314A with the 1100 chromatograph of liquid detectors that AGILENT produces, except that the retention time of fingerprint image because of the dead volume difference makes a difference, figure is similar substantially, as under the condition of control dead volume, same available this kind Instrument measuring, because the released state of chromatographic column, the energy of detector all are in dynamically, for the control of system suitability, estimate the most effective to use retinue system object of reference.Preparation about retinue system object of reference has three kinds, in the middle of existing further the investigation, tentatively drafts with lyophilizing as retinue system object of reference.Test with retinue system object of reference, the total chromatographic peak of appearance should be no less than with reference to collection of illustrative plates, and figure should be similar.Continuous sample introduction 5 pins, the similarity between its figure should be not less than 0.99, whether reaches balance fully with evaluation system.
2, sample treatment and assay method
Because sample is an injection, strict control is all arranged on clarity and microgranule, and keep the physical presence state of each material in the injection as far as possible, for simplifying the sample preparation process, the integrity of guarantee information, and can mate with mobile phase, the mode of injection direct injected adopted.Sample size 10 μ l.Assay method be aqueous chromatographic column-phosphate buffered solution (pH5.5)-isocratic elution be mobile phase as separation condition, a chromatogram is promptly finished separation determination.
3, data processing method
Generally with 1% as the related substance limit, set smallest peaks area should guarantee detecting greater than the chromatographic peak of total peak area 1.0% when the chemical drugs related substance detects.Less because of this product overall information amount, when integral parameter was set, the peak area that is about 0.4% chromatographic peak with the peak area percentage rate was made as the smallest peaks area value.Thresholding should guarantee to get rid of the information that the baseline noise effect can detect fingerprint peaks again.
4, similarity limit
Investigate the similarity of 16 batch samples and standard finger-print with Chinese Pharmacopoeia Commission's chromatographic fingerprints of Chinese materia medica similarity software, show that similarity all greater than 0.95, is higher than 0.9 standard.
(3) method validation of finger printing standard
1, repeatability checking
Because this standard adopts direct injected, the repeated experiment in the chromatographic condition research also can be considered the repeatability test, when by a people at same instrument, when different time was measured, the RSD of measurement result was 0.2%; When being measured at same laboratory by two people, the RSD of similarity is 0.5%.
2, ruggedness checking
In order to verify this standard in the different experiments chamber, use different instruments, the reviewer tests according to this standard by production unit, and assay and its similarity of this laboratory comparison are that RSD is 0.93%.
3, the globality evaluation of finger printing standard
In the research of expansion side and chromatographic condition, the research center of gravity is the expansion of chromatography component and detects, because we have adopted plurality of color spectral condition and expansion pattern method of deploying, its ionic medium is to (double ion)-gradient organic solvent method of development, high performance capillary electrophoresis, isoconcentration phosphate recoil solution-gradient organic solvent method of development all can launch chromatography component preferably, overcome the possibility that separating degree influences globality; Owing to be subjected to the restriction of unfolding condition, mass flow rate sensitive detector such as mass spectrum, evaporative light scattering detector etc. are all lack scope for their abilities, in order to solve chromatography component recall rate problem, once adopted specific function--(MAXPLOT) maximization of WATTERS company 996 detectors and work station, extracting the chromatogram of each chromatography component absorption maximum and the chromatogram of different wave length compares, when selecting 242nm for the detection wavelength, the recall rate of its chromatography component is 82% of a maximization chromatogram, be the highest testing conditions of recall rate, satisfied the principle that maximum information quantizes.
4, test sample stability test
The stability testing method of intending usefulness is for being reference standard with 0 month finger printing, and the similarity of more different rest period finger printing is estimated the total major constituent of placement sample with this and changed; When similarity was 1, promptly two figures were identical.Because chromatographic parameter mostly is dynamic change, even same sample cannot say for sure that also two chromatograms are in full accord, coefficient of similarity is fixed on be considered as unanimity more than 0.90, improve the credibility of evaluation " recall rate that total major constituent changes " with this.
| 0 month | January | | March | |
| Reference | ||||
| 1 | 0.999 | 0.998 | 0.998 |
The result shows: with 0 month finger printing was reference standard, the similarity of more different rest period finger printing, and all more than 0.95, expression has good stability.
With Herba Leonuri injection of the present invention is sample, and the liquid-phase chromatography method and the condition that adopt embodiment 4 to provide are measured result such as Fig. 4.Among the figure, S is sample actual measurement spectrogram of the present invention, and R is retinue object of reference sample actual measurement spectrogram.Interpretation of result shows that similarity is 0.997.
Claims (10)
1, a kind of preparation method of motherwort injection, it comprises the steps: adopting aqueous extraction-alcohol precipitation technology to make adding proper amount of active carbon and 0.5%-5% adsorbent in the medicinal liquid to Herba Leonuri, be mixed in deposit a period of time in the medicinal liquid after, filter, reclaim ethanol, add the injection water then and make to the desired concn specification, fine straining, embedding, sterilization, promptly.
2, according to the process of claim 1 wherein that the consumption of active carbon is the 0.01%-0.5w/v% of described amount of liquid medicine.
3, according to the method for claim 1 or 2, wherein, do not add benzyl alcohol in the preparation process.
4, according to arbitrary method of claim 1-3, wherein, described adsorbent is selected from one or more in the mineral of Talcum, bentonite, kieselguhr and potter's clay.
5, according to the method for claim 4, wherein, described adsorbent is that to contain the activatory weight ratio of sterilizing be 1: the Talcum of 0.1-10 and kieselguhr.
6,, wherein, when injection adopts spectrophotography to survey to contain total alkaloids in every 1ml injection and counts 20mg with stachydrine hydrochloride, adopt the HPLC method to measure hydrochloric stachydrine and be no less than 8mg according to the motherwort injection of either party's method of claim 1-5 preparation.
7, according to the injection of claim 6, wherein, the above microgranule of 10 μ m in the injection≤4000 contains the above microgranule of 25 μ m≤400.
8, according to the motherwort injection of claim 7, wherein, the injection product has following physicochemical property:
Protein is got this product 1ml, adds 30% sulfosalicylic acid test solution 1ml of new preparation, mixes, and places 5 minutes, does not occur muddy, up to specification;
Tannin is got this product 1ml, adds the physiological sodium chloride solution 5ml that contains 1% Ovum Gallus domesticus album of new preparation, places 10 minutes, does not occur muddy or precipitation, and is up to specification;
Resin is got this product 5ml, adds 1 of hydrochloric acid, places 30 minutes, precipitation do not occur, and is up to specification;
Oxalates: it is an amount of to get this product, regulates pH value to 1～2 with dilute hydrochloric acid, filters, and gets filtrate 2ml, and filtrate is regulated PH to 5～6, adds 2～3 of 3% calcium chloride solutions.Placed 10 minutes, and do not occur muddy or precipitation.
9, according to the motherwort injection of the arbitrary method preparation of claim 1-5, it is characterized in that this injection adopts Waters 2690 chromatograph of liquid; Chromatographic column: Aichrom Bond-AQ C184.6mm * 250mm, 5 μ m; Mobile phase: 0.1mol/L sodium dihydrogen phosphate; Column temperature: 30 ℃; Wavelength: 242nm measures down, the chromatographic peak of appearance be not less than 0.9 as Fig. 1 spectrogram shape similarity.
10, injection according to Claim 8, wherein, chromatographic peak that this injection determines and described collection of illustrative plates figure similarity are not less than 0.95.
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| CN2006101502848A CN1973854B (en) | 2006-10-16 | 2006-10-16 | Process of preparing motherwort injection |
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| CN2006101502848A CN1973854B (en) | 2006-10-16 | 2006-10-16 | Process of preparing motherwort injection |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102846704A (en) * | 2011-06-27 | 2013-01-02 | 西安千禾药业有限责任公司 | A Leonurus japonicus injection, its preparation method, and method for detecting total alkaloids |
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2006
- 2006-10-16 CN CN2006101502848A patent/CN1973854B/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102846704A (en) * | 2011-06-27 | 2013-01-02 | 西安千禾药业有限责任公司 | A Leonurus japonicus injection, its preparation method, and method for detecting total alkaloids |
| CN102846704B (en) * | 2011-06-27 | 2014-09-17 | 西安千禾药业有限责任公司 | A Leonurus japonicus injection, its preparation method, and method for detecting total alkaloids |
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| CN1973854B (en) | 2011-06-08 |
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