CN101982189A - Method for detecting salvia heart-soothing capsules - Google Patents
Method for detecting salvia heart-soothing capsules Download PDFInfo
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Abstract
The invention provides a method for detecting the quality of salvia heart-soothing capsules. The method comprises all or partial items of character detection, identification, inspection and content measurement, wherein the character is capsules which are filled with dark brown powder, and have mild and subastringent taste; the identification means to use salvia contrast medicament as contrast, and a thin-layer chromatography method is adopted for salvia qualitative identification; the inspection needs to accord with regulations related to capsule item in the appendix IL of Chinese pharmacopoeia 2010 (vol.1); and the content measurement is to detect the content of main components tanshinone IIA and salvianolic acid B of the capsules by adopting high-efficiency liquid chromatography method. The quality detecting method has the advantages of strong specificity, high accuracy, good stability, good reproducibility, high reclamation rate, accurate measurement and simpleness and convenience, measures the contents of water-soluble ingredients and ethanol soluble ingredients of the capsules, improves the quality control standard of the salvia heart-soothing capsules, and can effectively control fake salvia heart-soothing capsule production of illegal manufacturers or manufacturers with low level of process control technology so as to ensure the clinical curative effect of the preparation.
Description
Technical field
The present invention relates to a kind of detection method of Danshen heart-benefiting pharmaceutical capsules, belong to the technical field of medicine being carried out quality control.
Background technology
The Danshen heart-benefiting capsule is the 7th kind of recording of " the Sanitation Ministry medicine standard " Chinese traditional patent formulation preparation, and standard is numbered WS3-B-1294-93, write out a prescription to the red rooted salvia extract of Chinese medicine simply, and be the capsule of pure Chinese medicine.It has blood circulation promoting and blood stasis dispelling, and the effect of tranquillizing and allaying excitement is used for the treatment of the angina pectoris that coronary heart disease causes clinically, and disease such as uncomfortable in chest and cardiopalmus is to be used for the treatment of the angina pectoris that coronary heart disease causes in the market, the common drug of disease such as uncomfortable in chest and cardiopalmus.But existing quality control standard is simple, product quality is wayward, serve as that the thin layer chromatography discriminating has been carried out in contrast with Tanshinone I I A reference substance just to the Danshen heart-benefiting pharmaceutical capsules, and Main Ingredients and Appearance Tanshinone I I A wherein, the content of salvianolic acid B are not measured, discrimination method wherein is not only loaded down with trivial details but also can't reflect Main Ingredients and Appearance Tanshinone I I A, salvianolic acid B of Radix Salviae Miltiorrhizae etc. comprehensively.Because Danshen heart-benefiting capsule good effect, consumption is big, so the consumption of red rooted salvia is corresponding very big, other many medicines also will be used Radix Salviae Miltiorrhizae, and resource-constrained, so the price of red rooted salvia is also very high.Therefore, part illegal manufacturer having occurred throws Radix Salviae Miltiorrhizae less or contains the partly plant extract of the composition production that feeds intake of Radix Salviae Miltiorrhizae with the Radix Salviae Miltiorrhizae extract that only contains red sage root component partly or other when producing the Danshen heart-benefiting capsule; In addition, existing pure soluble constituent of the active ingredient of Radix Salviae Miltiorrhizae such as Tanshinone I I A have water-soluble components such as salvianolic acid B again, and these two kinds of compositions are less stable all, production technology is not noted controlling by the manufacturer that has, can make wherein partly in addition all active ingredient incur loss; Above problem has had a strong impact on the clinical efficacy of said preparation.
Summary of the invention
The objective of the invention is to, provide a kind of Danshen heart-benefiting capsular quality determining method.It is simple to the present invention is directed to existing quality control standard, the uppity shortcoming of product quality, content, characteristic and preparation technology thereof according to red rooted salvia among the we and extract thereof, research has customized feasible discriminating and content assaying method, controlling the capsular quality of Danshen heart-benefiting effectively, thereby guarantee the clinical efficacy of said preparation.
Danshen heart-benefiting capsule of the present invention is to constitute like this: it is to be added adjuvants such as starch, Pulvis Talci, magnesium stearate to be prepared from by the red rooted salvia extract.
The capsular preparation method of Danshen heart-benefiting is: the Radix Salviae Miltiorrhizae behind the decocting of learning from else's experience, and drying is pulverized, and adds 90% alcohol reflux three times, 2 hours for the first time, second and third time each 1.5 hours filters respectively, merging filtrate, and concentrating under reduced pressure becomes extractum, the ordinary water that adds 10 times of amounts of extractum, the limit edged stirs, and leaves standstill, and filters, the precipitate cold drying is pulverized, and sieves, and promptly gets Radix Salviae Miltiorrhizae extract, get Radix Salviae Miltiorrhizae extract 200g, add starch 88g, Pulvis Talci 10g, magnesium stearate 2g, mixing incapsulates, and makes 1000, promptly.
Detection method of the present invention mainly comprise in character, discriminating, inspection, the assay project partly or entirely:
Discriminating is to be contrast with the Radix Salviae Miltiorrhizae control medicinal material, adopts thin layer chromatography to differentiate; Assay is that wherein the Main Ingredients and Appearance Tanshinone I I A and the content of salvianolic acid B are measured.
The composition discrimination method is:
Get this product content 2～4g, porphyrize, the 5～10ml that adds diethyl ether, jolting was placed 40～80 minutes, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1～2ml makes dissolving, as need testing solution.Get Radix Salviae Miltiorrhizae control medicinal material 0.5～1.5g, porphyrize, the 5～10ml that adds diethyl ether, jolting was placed 40～80 minutes, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1～2ml makes dissolving, in contrast medical material solution.According to an appendix VI of Chinese Pharmacopoeia version in 2010 B thin layer chromatography, draw each 3～10 μ l of above-mentioned two kinds of solution, putting respectively on same silica gel g thin-layer plate, is developing solvent, wherein benzene with benzene and ethyl acetate: ethyl acetate=18～20: 1～1.5, launch, take out, dry, in the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color;
Content assaying method is:
The content assaying method of Tanshinone I I A:
According to an appendix VI of Chinese Pharmacopoeia version in 2010 D high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; Methanol: water=70～80: 20～30 is mobile phase; The detection wavelength is 268～272nm; Number of theoretical plate calculates by Tanshinone I I A peak should be not less than 2000; Precision takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shake up, precision is measured 2ml, puts in the brown measuring bottle of 25ml, add methanol to scale, shake up, promptly get the reference substance solution that contains tanshinone 16 μ g among every 1ml; get this product content 0.7g, porphyrize, accurate claim fixed; put in the tool plug conical flask; the accurate methanol 50ml that adds, claim decide weight, through power 120W, frequency 59KHZ supersound process 15～25 minutes; put cold; close plug, claim again to decide weight, supply with methanol and subtract weight loss; shake up; filtration, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains Tanshinone I I A must not be less than 0.6mg;
The content assaying method of salvianolic acid B:
According to an appendix VI of Chinese Pharmacopoeia version in 2010 D high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; With methanol: acetonitrile: formic acid: water=25～35: 8～10: 0.5～1.5: 57～61 is mobile phase; The detection wavelength is 284～288nm; Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000; It is an amount of that precision takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly gets reference substance solution; get this product content 0.6g, porphyrize, accurate claim fixed; put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims decide weight; through power 120W, frequency 59KHZ supersound process 15～25 minutes, take out, put cold; claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up; filtration, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains salvianolic acid B must not be less than 6mg.
Detection method of the present invention comprise following partly or entirely:
Character: this product is a capsule, and content is the dark-brown powder; Lightly seasoned, little puckery;
Differentiate: get this product content 3g, porphyrize, 5ml adds diethyl ether, jolting was placed 1 hour, filtered, filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution, get Radix Salviae Miltiorrhizae control medicinal material 1g, porphyrize, 5ml adds diethyl ether, jolting was placed 1 hour, filtered, filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, in contrast medical material solution.According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene: ethyl acetate=19: 1 is developing solvent, launches, and takes out, dry, in the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color;
Check: should meet relevant every regulation under an appendix IL of Chinese Pharmacopoeia version in 2010 the capsule item;
Assay:
The content assaying method of Tanshinone I I A:, be filler with octadecylsilane chemically bonded silica according to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography; Methanol: water=75: 25 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the tanshinone peak should be not less than 2000; Precision takes by weighing tanshinone reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets the reference substance solution that contains tanshinone 16 μ g among every 1ml; Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, put cold, close plug, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains tanshinone must not be less than 0.6mg.
The content assaying method of salvianolic acid B is:
According to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; With methanol: acetonitrile: formic acid: water=30: 10: 1: 59 is mobile phase; The detection wavelength is 286nm; Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000; It is an amount of that precision takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly gets reference substance solution; Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, take out, put cold, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains salvianolic acid B must not be less than 6mg.
Discriminating can also be adopted following method:
Get the content of 1 of this product, the 5ml that adds diethyl ether, jolting was placed 1 hour, filter, filtrate is concentrated into about 1ml, and it is an amount of to add neutral alumina, mixes thoroughly, a neutral alumina post that fills in advance (the 8g internal diameter 15mn) top of packing into after the drying, with acetic acid second vinegar eluting, collect effusive at first eluent 1ml, as need testing solution.Other gets the tanshinone reference substance, adds ethyl acetate and makes the solution that every 1ml contains 2mg, in contrast product solution.According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, selecting respectively on same silica gel g thin-layer plate with normal hexane-acetic acid second vinegar (17: 3) be developing solvent, launches, taking-up is dried.In the test sample chromatograph, with contrast chromatograph corresponding position on, show the speckle of identical color.
For guaranteeing detection method science of the present invention, reasonable, feasible, the applicant has carried out a series of experimentation, and concrete experimental data is as follows:
One, thin layer chromatography is differentiated:
Because this product is the red rooted salvia extract, whole active ingredients of crude drug have been kept, in order to check this product whether to keep whole active ingredients of crude drug, simple control with a certain or two kinds of compositions is not all right, so select for use the Radix Salviae Miltiorrhizae control medicinal material to contrast, differentiate with thin layer chromatography.We have done a large amount of tests for the selection of developing solvent and lamellae.Directly point sample is on silica gel g thin-layer plate, it is more satisfactory to launch the back effect, but different its separating degree differences of developing solvent, so investigated several development systems, from simple to complexity, benzene is arranged: ethyl acetate=10: 5, benzene: ethyl acetate=10: 1, be with benzene at last: ethyl acetate=19: 1 is developing solvent, and separating degree is good, clear spot launches effective.So following method is adopted in decision:
Get this product content 3g, porphyrize, 5ml adds diethyl ether, jolting was placed 1 hour, filtered, filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution, get Radix Salviae Miltiorrhizae control medicinal material 1g, porphyrize, 5ml adds diethyl ether, jolting was placed 1 hour, filtered, filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, in contrast medical material solution.According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene: ethyl acetate=19: 1 is developing solvent, launches, and takes out, dry, in the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color.
Two, the content assaying method of tanshinone:
According to high effective liquid chromatography for measuring:
The selection of index components: with reference to the analysis of components of Radix Salviae Miltiorrhizae, tanshinone is that it mainly contains one of effective constituent, is main pure soluble constituent, so selected tanshinone is one of index composition, carry out the control of finished product content, adopt high performance liquid chromatography the capable assay of tanshinone.
1. instrument and reagent
SHIMADZU SPD-10AVP type high performance liquid chromatograph, UV762 type dual-beam ultraviolet spectrophotometer, FA2004 electronic analytical balance.Methanol is chromatographically pure, and water is double distilled water, and all the other reagent are analytical pure, and reference substance is provided by middle inspection.
2. detect the selection of wavelength
Use for reference Tanshinone I I A content assaying method under red rooted salvia item of Chinese Pharmacopoeia version in 2010.Select identical detection wavelength, be 270nm, detect with UV-detector.And by following experimental verification: get the methanol solution of Tanshinone I I A reference substance, the place carries out sweep measuring at 200～600nm wavelength, and it is consistent that maximum absorption wavelength is measured wavelength (270nm) with pharmacopeia Tanshinone I I A.
3. the selection of mobile phase
With reference to tanshinone content assaying method under red rooted salvia item of Chinese Pharmacopoeia version in 2010 this product is carried out assay, promptly select methanol: water=75: 25 is mobile phase.
Chromatographic column: octadecylsilane chemically bonded silica is a filler.
Flow velocity: 1.0ml/min.
Column temperature: 28 ℃.
4. the preparation of test solution
4.1 the preparation of reference substance solution
Precision takes by weighing tanshinone reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets the reference substance solution that contains tanshinone 16 μ g among every 1ml;
4.2 the preparation of sample solution
Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, put cold, close plug, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution;
4.3 extract the investigation of solvent
Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, add 50% methanol, methanol, each 50ml of ethanol respectively, claim to decide weight, supersound process (power 120W, frequency 59KHZ) 20 minutes is put cold, close plug claims to decide weight again, supplies with coordinative solvent to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Measure by above-mentioned chromatographic condition, calculate, the results are shown in Table 1.
Table 1 extracts solvent and investigates the result
| Extract solvent | 50% methanol | Methanol | Ethanol |
| Tanshinone content (mg/ml) | 0.038 | 0.041 | 0.037 |
As shown in Table 1, be to extract solvent with methanol, extraction efficiency is better than 50% methanol, ethanol, and therefore selecting methanol for use is to extract solvent.
4.4 the investigation of extracting method
Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, a supersound process (power 120W, frequency 59KHZ) 20 minutes, another part were put in the water-bath reflux 20 minutes, put coldly, close plug claims to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Measure by above-mentioned chromatographic condition, calculate, the results are shown in Table 2.
Table 2 extracting method is investigated the result
| Extracting method | Reflux, extract, | Supersound extraction |
| Tanshinone content (mg/ml) | 0.040 | 0.041 |
As shown in Table 2, two kinds of methods are extracted several zero differences of content, select for use convenient and simple supersound process (power 120W, frequency 59KHZ) to extract more than therefore.
4.5 the investigation of extraction time
Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, supersound process (power 120W, frequency 59KHZ) is 20 minutes, 30 minutes, 40 minutes respectively, puts cold, close plug claims to decide weight again, supplies with methanol to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Measure by above-mentioned chromatographic condition, calculate, the results are shown in Table 3.
Table 3 extraction time investigation result
| Extraction time (minute) | 20 | 30 | 40 |
| Tanshinone content (mg/ml) | 0.040 | 0.041 | 0.040 |
As shown in Table 3, several extraction times all can extract more fully, several zero differences of the result of assay, so extraction time be chosen as 20 minutes.
In sum, the preparation method of need testing solution is: get this product content 0.7g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, claim to decide weight, supersound process (power 120W, frequency 59KHZ) 20 minutes, put coldly, close plug claims to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution.
5. serviceability test
5.1 stability test is got this product content 0.7g, porphyrize, and accurate the title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, supersound process (power 120W, frequency 59KHZ) 20 minutes, put cold, close plug, weight decided in title again, supplies with methanol to subtract weight loss, shakes up, filter, get subsequent filtrate, preserve under the room temperature, get reference substance solution and need testing solution respectively at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, measure, the results are shown in Table 4 by the above content assaying method of drafting.
Table 4 stability test result
| Standing time (H) | 0 | 2 | 4 | 6 | 8 |
| Content (mg/ml) | 0.0401 | 0.0400 | 0.0401 | 0.0399 | 0.0400 |
As shown in Table 4, reference substance solution and need testing solution are measured in back 8 hours of preparation, and the result is stable.
5.2 the mobile phase of different proportion
Experiment is once with methanol: water=70: 25, methanol: water=75: 25, methanol: water=75: 20 is mobile phase, and flow velocity: 1.0ml/min is with SHIMADZUC
18Post (5 μ m, 4.6 * 250mm) is analytical column, 28 ℃ of column temperatures, research experiment all can reach satisfied separation, and measurement result is respectively 0.0403,0.0409,0.0396, and RSD is 1.61%.
5.3 different chromatography post
SHIMADZU C was once used in experiment
18Post (5 μ m, 4.6 * 250mm), the ZIVCHROMI post (5 μ, 4.6 * 150mm) with Kromsil C
18Post (5 μ m, 46 * 150mm) is analytical column, with methanol: water=75: 25 is mobile phase, and measurement result is respectively 0.0408,0.0412,0.0399, and RSD is 1.64%.
In sum, the ruggedness of this method is better.
The ratio of mobile phase changes slightly, and tanshinone and other components all can reach baseline separation, and system suitability is good.Octadecylsilane chemically bonded silica with SHIMADZU, ZIVCHROMI and three kinds of brands of Kromsil is the chromatographic column of filler respectively, and tanshinone and other components all can reach baseline separation, and system suitability is good.
6. content assaying method is learned research
6.1 the standard curve and the range of linearity
Precision takes by weighing tanshinone reference substance 10.2986mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured this solution 1ml, 3ml, 5ml, 7ml, 9ml respectively, puts in the 50ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, promptly.Tanshinone is respectively 4.11944ug, 12.35832ug, 20.59720ug, 28.83608ug, 37.07496ug, as need testing solution.Respectively accurate each the 10 μ l of above-mentioned need testing solution that draw draft chromatographic condition analysis by above, measure peak area, the results are shown in Table 5.
The table 5 tanshinone reference substance range of linearity is investigated
Tanshinone I I A concentration is good linear relationship with peak area in 4.11944～37.07496 μ g/ml scopes, regression equation and correlation coefficient: A=295.2328C-79.3553, γ=0.9997 (C is a tanshinone concentration, and the μ g/ml of unit, A are peak area).
6.2 replica test
By the content assaying method of drafting, totally 6 parts of sample thiefs prepare need testing solution respectively, measure by above-mentioned chromatographic condition, the results are shown in Table 6.
Table 6 replica test result
As shown in Table 6, RSD=0.508% shows that repeatability is good.
6.3 precision
From with batch this product sample, take a sample 5 parts simultaneously, by content assaying method of the present invention, prepare the sample test liquid respectively and measure.The content of tabulation statistics various kinds and average content, RSD.
Table 7 precision is investigated the result
From last table 7 as can be known, its precision is better.
6.4 sample determination
According to the assay method of drafting the tanshinone content of three batch samples (lot number is 100701,100702,100703) is measured, the every batch of parallel assay 2 times the results are shown in Table 8.
Tanshinone assay result in table 8 sample
Three, the content assaying method of salvianolic acid B:
According to high effective liquid chromatography for measuring:
The selection of index components: with reference to the analysis of components of Radix Salviae Miltiorrhizae, salvianolic acid B is that it mainly contains one of effective constituent, is main water-soluble components, so our selected salvianolic acid B is one of index composition, carries out the control of finished product content.Adopt high performance liquid chromatography that salvianolic acid B is carried out assay.
1. instrument and reagent
SHIMADZU SPD-10AVP type high performance liquid chromatograph, UV762 type dual-beam ultraviolet spectrophotometer, FA2004 electronic analytical balance.Methanol is chromatographically pure, and water is double distilled water, and all the other reagent are analytical pure, and reference substance is provided by middle inspection.
2. detect the selection of wavelength
Use for reference the content assaying method of salvianolic acid B under red rooted salvia item of Chinese Pharmacopoeia version in 2010.Select identical detection wavelength, be 286nm, detect with UV-detector.And by following experimental verification: get the methanol solution of salvianolic acid B reference substance, the place carries out sweep measuring at 200～600nm wavelength, and maximum absorption wavelength is consistent with the mensuration wavelength (286nm) of pharmacopeia salvianolic acid B.
3. the selection of mobile phase
Content assaying method with reference to salvianolic acid B under red rooted salvia item of Chinese Pharmacopoeia version in 2010 carries out assay to this product, promptly selects methanol: acetonitrile: formic acid: water=30: 10: 1: 59 are mobile phase;
Chromatographic column: octadecylsilane chemically bonded silica is a filler;
Flow velocity: 1.0ml/min;
Column temperature: 26 ℃.
4. the preparation of test solution
4.1 the preparation of reference substance solution
It is an amount of to get the salvianolic acid B reference substance, adds 75% methanol and makes the solution that every 1ml contains 0.14mg, promptly gets reference substance solution;
4.2 the preparation of sample solution
Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, take out, put cold, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution.
4.3 extract the investigation of solvent
Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, accurate 50% methanol, 75% methanol, the methanol 50ml of adding claims to decide weight, supersound process (power 120W, frequency 59KHZ) 20 minutes is taken out, put coldly, claim again to decide weight, supply the weight that subtracts mistake with coordinative solvent, shake up, filter, get subsequent filtrate, promptly get need testing solution; Measure by above-mentioned chromatographic condition, calculate, the results are shown in Table 9.
Table 9 extracts solvent and investigates the result
| Extract solvent | 50% methanol | 75% methanol | Methanol |
| Content of danshinolic acid B (mg/ml) | 0.511 | 0.518 | 0.492 |
As shown in Table 9, serve as to extract solvent with 75% methanol, extraction efficiency is better than 50% methanol, methanol, therefore selects for use 75% methanol for extracting solvent.
4.4 the research of extracting method
Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, a supersound process (power 120W, frequency 59KHZ) 20 minutes, another part were put in the water-bath reflux 20 minutes, take out, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Measure by above-mentioned chromatographic condition, calculate, the results are shown in Table 10.
Table 10 extracting method is investigated the result
| Extracting method | Reflux, extract, | Supersound extraction |
| Content of danshinolic acid B (mg/ml) | 0.516 | 0.517 |
As shown in Table 10, two kinds of methods are extracted several zero differences of content, select for use convenient and simple supersound process (power 120W, frequency 59KHZ) to extract more than therefore.
4.5 the investigation of extraction time
Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, supersound process (power 120W, frequency 59KHZ) 10 minutes, 20 minutes, 30 minutes is taken out, put coldly, claim again to decide weight, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Measure by above-mentioned chromatographic condition, calculate, the results are shown in Table 11.
Table 11 extraction time investigation result
| Extraction time (minute) | 10 | 20 | 30 |
| Content of danshinolic acid B (mg/ml) | 0.502 | 0.518 | 0.518 |
As shown in Table 11, several extraction times all can extract more fully, and the result difference of assay is little, do not have difference in 20 minutes and 30 minutes, so extraction time are chosen as 20 minutes.
In sum, the preparation method of need testing solution is: get this product content 0.6g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask, the accurate 75% methanol 50ml that adds, claim to decide weight, supersound process (power 120W, frequency 59KHZ) 20 minutes, take out, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution.
5. serviceability test
5.1 stability test
Get this product content 0.6g, porphyrize, the accurate title, decide, and puts in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, supersound process (power 120W, frequency 59KHZ) 20 minutes, take out, put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with 75% methanol, filter, get subsequent filtrate, preserve under the room temperature, get reference substance solution and need testing solution respectively at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, press content assaying method and measure, the results are shown in Table 12.
Table 12 stability test result
| Standing time (H) | 0 | 2 | 4 | 6 | 8 |
| Content (mg/ml) | 0.517 | 0.516 | 0.518 | 0.518 | 0.516 |
As shown in Table 12, reference substance solution and need testing solution are measured in back 8 hours of preparation, and the result is stable.
5.2 the mobile phase of different proportion
Experiment is once with methanol: acetonitrile: formic acid: water=30: 10: 2: 58, methanol: acetonitrile: formic acid: water=25: 15: 1: 59, methanol: acetonitrile: formic acid: water=30: 10: 1: 59 is mobile phase, and flow velocity: 1.0ml/min is with SHIMADZUC
18Post (5 μ m, 4.6 * 250mm) is analytical column, 26 ℃ of column temperatures, research experiment all can reach satisfied separation, and measurement result is respectively 0.519,0.517,0.516, and RSD is 0.29%.
5.3 different chromatography post
SHIMADZU C was once used in experiment
18Post (5 μ m, 4.6 * 250mm), the ZIVCHROMI post (5 μ, 4.6 * 150mm) with Kromsil C
18Post (5 μ m, 4.6 * 150mm) is analytical column, with methanol: acetonitrile: formic acid: water=30: 10: 1: 59 is mobile phase, and measurement result is respectively 0.506,0.514,0.519, and RSD is 1.28%.
In sum, the ruggedness of this method is better.
The ratio of mobile phase changes slightly, and salvianolic acid B and other components all can reach baseline separation, and system suitability is good.Octadecylsilane chemically bonded silica with SHIMADZU, ZIVCHROMI and three kinds of brands of Kromsil is the chromatographic column of filler respectively, and salvianolic acid B and other components all can reach baseline separation, and system suitability is good.
6. content assaying method is learned research
6.1 the standard curve and the range of linearity
Precision takes by weighing salvianolic acid B reference substance 28.3147mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured this solution 1ml, 3ml, 5ml, 7ml, 9ml respectively, puts in the 50ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, promptly.Salvianolic acid B is respectively 11.32588ug, 33.97764ug, 56.6294ug, 79.28116ug, 101.93292ug, as need testing solution.Respectively accurate each the 10 μ l of above-mentioned need testing solution that draw draft chromatographic condition analysis by above, measure peak area, the results are shown in Table 13.
The table 13 salvianolic acid B reference substance range of linearity is investigated
| Reference substance concentration C (μ g/ml) | ?11.32588 | 33.97764 | 56.6294 | 79.28116 | 101.93292 |
| Peak area A | 535949 | 1607917 | 2679786 | 3751541 | 4823517 |
Salvianolic acid B concentration is good linear relationship with peak area in 11.32588～101.93292 μ g/ml scopes, regression equation and correlation coefficient: A=473.213C-144.0953, γ=0.9998 (C is a salvianolic acid B concentration, and the μ g/ml of unit, A are peak area).
6.2 replica test
By content assaying method of the present invention, totally 6 parts of sample thiefs prepare need testing solution respectively, measure by above-mentioned chromatographic condition, the results are shown in Table 14.
Table 14 replica test result
As shown in Table 14, RSD=1.71% shows that repeatability is good.
6.3 precision
From with batch this product sample, take a sample 5 parts simultaneously, by content assaying method of the present invention, prepare the sample test liquid respectively and measure.The content of tabulation statistics various kinds and average content, RSD.
Table 15 precision is investigated the result
From last table 15 as can be known, its precision is better.
6.4 sample determination
According to the assay method of drafting the content of danshinolic acid B of three batch samples (lot number is 091001,091002,091003) is measured, the every batch of parallel assay 2 times the results are shown in Table 16.
Content of danshinolic acid B measurement result in the table 16
Beneficial effect of the present invention:
Compared with prior art, the present invention differentiates and assay by above thin layer chromatography, discriminating and assay method that clear and definite quality index and each quality index have been arranged, these methodological sciences are reasonable, practical, simple and convenient, specificity is strong, the precision height, good stability, favorable reproducibility, response rate height, measurement result is accurate, and water-soluble components and pure soluble constituent wherein all carried out assay, improve the capsular quality control standard of Danshen heart-benefiting, can control illegal manufacturer effectively or the low manufacturer of process control technology level produces Danshen heart-benefiting capsule of poor quality, thereby guaranteed the clinical efficacy of said preparation.
The specific embodiment
Embodiment 1: the capsular detection method of Danshen heart-benefiting
Character: this product is a capsule, and content is the dark-brown powder; Lightly seasoned, little puckery.
Differentiate: get this product content 3g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution; Get Radix Salviae Miltiorrhizae control medicinal material 1g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, in contrast medical material solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene: ethyl acetate=19: 1 be developing solvent, launches, and taking-up is dried; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color.
Check: should meet relevant every regulation under an appendix IL of Chinese Pharmacopoeia version in 2010 the capsule item.
Assay:
The content assaying method of tanshinone:, be filler with octadecylsilane chemically bonded silica according to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography; Methanol: water=75: 25 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the tanshinone peak should be not less than 2000; Precision takes by weighing tanshinone reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets the reference substance solution that contains tanshinone 16 μ g among every 1ml; Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, put cold, close plug, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains Tanshinone I IA must not be less than 0.6mg.
Assay:
The content assaying method of salvianolic acid B is:
According to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; With methanol: acetonitrile: formic acid: water=30: 10: 1: 59 is mobile phase; The detection wavelength is 286nm; Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000; It is an amount of that precision takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly gets reference substance solution; Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, take out, put cold, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains salvianolic acid B must not be less than 6mg.
Embodiment 2: the capsular detection method of Danshen heart-benefiting
Character: this product is a capsule, and content is the dark-brown powder; Lightly seasoned, little puckery.
Differentiate: get this product content 3g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution; Get Radix Salviae Miltiorrhizae control medicinal material 1g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, in contrast medical material solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene: ethyl acetate=19: 1 be developing solvent, launches, and taking-up is dried; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color.
Check: should meet relevant every regulation under an appendix IL of Chinese Pharmacopoeia version in 2010 the capsule item.
Assay:
The assay of tanshinone:, be filler with octadecylsilane chemically bonded silica according to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography; Methanol: water=75: 25 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the tanshinone peak should be not less than 2000; Precision takes by weighing tanshinone reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets the reference substance solution that contains tanshinone 16 μ g among every 1ml; Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, put cold, close plug, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains tanshinone must not be less than 0.6mg;
Embodiment 3: the capsular detection method of Danshen heart-benefiting
Character: this product is a capsule, and content is the dark-brown powder; Lightly seasoned, little puckery.
Differentiate: get this product content 3g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution; Get Radix Salviae Miltiorrhizae control medicinal material 1g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, in contrast medical material solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene: ethyl acetate=19: 1 be developing solvent, launches, and taking-up is dried; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color.
Check: should meet relevant every regulation under an appendix IL of Chinese Pharmacopoeia version in 2010 the capsule item.
Assay:
The assay of salvianolic acid B:, be filler with octadecylsilane chemically bonded silica according to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography; With methanol: acetonitrile: formic acid: water=30: 10: 1: 59 is mobile phase; The detection wavelength is 286nm; Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000; It is an amount of that precision takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly gets reference substance solution; Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, take out, put cold, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains salvianolic acid B must not be less than 6mg.
Embodiment 4: the capsular detection method of Danshen heart-benefiting
Character: this product is a capsule, and content is the dark-brown powder; Lightly seasoned, little puckery.
Differentiate: get this product content 2g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution; Get Radix Salviae Miltiorrhizae control medicinal material 0.5g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, in contrast medical material solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene: ethyl acetate=19: 1 be developing solvent, launches, and taking-up is dried; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color.
Check: should meet relevant every regulation under an appendix IL of Chinese Pharmacopoeia version in 2010 the capsule item.
Assay:
The content assaying method of tanshinone:, be filler with octadecylsilane chemically bonded silica according to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography; Methanol: water=70: 20 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the tanshinone peak should be not less than 2000; Precision takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets the reference substance solution that contains tanshinone 16 μ g among every 1ml; Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 25 minutes, put cold, close plug, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains tanshinone must not be less than 0.6mg.
Assay:
The content assaying method of salvianolic acid B is:
According to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; With methanol: acetonitrile: formic acid: water=25: 8: 0.5: 57 is mobile phase; The detection wavelength is 286nm; Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000; It is an amount of that precision takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly gets reference substance solution; Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, take out, put cold, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains salvianolic acid B must not be less than 6mg.
Embodiment 5: the capsular detection method of Danshen heart-benefiting
Character: this product is a capsule, and content is the dark-brown powder; Lightly seasoned, little puckery.
Differentiate: (1) gets this product content 3g, porphyrize, and the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution; Get Radix Salviae Miltiorrhizae control medicinal material 1g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, in contrast medical material solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene: ethyl acetate=19: 1 be developing solvent, launches, and taking-up is dried; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color.
(2) get the content of 1 of this product, the 5ml that adds diethyl ether, jolting was placed 1 hour, filter, filtrate is concentrated into about 1ml, and it is an amount of to add neutral alumina, mixes thoroughly, a neutral alumina post that fills in advance (the 8g internal diameter 15mn) top of packing into after the drying, with acetic acid second vinegar eluting, collect effusive at first eluent 1ml, as need testing solution.Other gets the tanshinone reference substance, adds ethyl acetate and makes the solution that every 1ml contains 2mg, in contrast product solution.According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, selecting respectively on same silica gel g thin-layer plate with normal hexane-acetic acid second vinegar (17: 3) be developing solvent, launches, taking-up is dried.In the test sample chromatograph, with contrast chromatograph corresponding position on, show the speckle of identical color.
Check: should meet relevant every regulation under an appendix IL of Chinese Pharmacopoeia version in 2010 the capsule item.
Assay:
The content assaying method of tanshinone:, be filler with octadecylsilane chemically bonded silica according to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography; Methanol: water=75: 25 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the tanshinone peak should be not less than 2000; Precision takes by weighing tanshinone reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets the reference substance solution that contains tanshinone 16 μ g among every 1ml; Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, put cold, close plug, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains tanshinone must not be less than 0.6mg.
Assay:
The content assaying method of salvianolic acid B is:
According to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; With methanol: acetonitrile: formic acid: water=30: 10: 1: 59 is mobile phase; The detection wavelength is 286nm; Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000; It is an amount of that precision takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly gets reference substance solution; Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, take out, put cold, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains salvianolic acid B must not be less than 6mg.
Claims (2)
1. capsular detection method of Danshen heart-benefiting, described Danshen heart-benefiting capsule are that the red rooted salvia extract adds adjuvants such as starch, Pulvis Talci, magnesium stearate and is prepared from, and described detection method comprises character, discriminating and inspection item; It is characterized in that: described detection method also comprises assay, and this assay is the assay to tanshinone and/or salvianolic acid B, and the assay of tanshinone is to be reference substance with the tanshinone, adopts high performance liquid chromatography to measure; The assay of salvianolic acid B is to be reference substance with the salvianolic acid B, adopts high performance liquid chromatography to measure;
Described discriminating is to be reference substance with the red rooted salvia, adopts thin layer chromatography to differentiate that discrimination method is:
Get this product content 2～4g, porphyrize, the 5～10ml that adds diethyl ether, jolting was placed 40～80 minutes, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1～2ml makes dissolving, as need testing solution; Get Radix Salviae Miltiorrhizae control medicinal material 0.5～1.5g, porphyrize, the 5～10ml that adds diethyl ether, jolting was placed 40～80 minutes, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1～2ml makes dissolving, in contrast medical material solution; According to an appendix VI of Chinese Pharmacopoeia version in 2010 B thin layer chromatography, draw each 3～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene and ethyl acetate is developing solvent, wherein benzene: ethyl acetate=18～20: 1～1.5, launch, take out, dry; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color;
The content assaying method of Tanshinone I I A is:
According to an appendix VI of Chinese Pharmacopoeia version in 2010 D high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; Methanol: water=70～80: 20～30 is mobile phase; The detection wavelength is 268～272nm; Number of theoretical plate calculates by the tanshinone peak and is not less than 2000; Precision takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shake up, precision is measured 2ml, puts in the brown measuring bottle of 25ml, add methanol to scale, shake up, promptly get the reference substance solution that contains tanshinone 16 μ g among every 1ml; get this product content 0.7g, porphyrize, accurate claim fixed; put in the tool plug conical flask; the accurate methanol 50ml that adds, claim decide weight, through power 120W, frequency 59KHZ supersound process 15～25 minutes; put cold; close plug, claim again to decide weight, supply with methanol and subtract weight loss; shake up; filtration, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains Tanshinone I I A must not be less than 0.6mg;
The content assaying method of salvianolic acid B is:
According to an appendix VI of Chinese Pharmacopoeia version in 2010 D high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; With methanol: acetonitrile: formic acid: water=25～35: 8～10: 0.5～1.5: 57～61 is mobile phase; The detection wavelength is 284～288nm; Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000; It is an amount of that precision takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly gets reference substance solution; get this product content 0.6g, porphyrize, accurate claim fixed; put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims decide weight; through power 120W, frequency 59KHZ supersound process 15～25 minutes, take out, put cold; claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up; filtration, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains salvianolic acid B must not be less than 6mg.
2. the capsular detection method of Danshen heart-benefiting according to claim 1, it is characterized in that: described detection method comprises:
Character: this product is a capsule, and content is the dark-brown powder; Lightly seasoned, little puckery;
Differentiate: get this product content 3g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution; Get Radix Salviae Miltiorrhizae control medicinal material 1g, porphyrize, the 5ml that adds diethyl ether, jolting was placed 1 hour, filtered, and filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, in contrast medical material solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2010 thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene: ethyl acetate=19: 1 be developing solvent, launches, and taking-up is dried; In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the speckle of same color;
Check: should meet relevant every regulation under an appendix IL of Chinese Pharmacopoeia version in 2010 the capsule item;
Assay:
The content assaying method of Tanshinone I I A:, be filler with octadecylsilane chemically bonded silica according to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography; Methanol: water=75: 25 is mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the tanshinone peak should be not less than 2000; Precision takes by weighing Tanshinone I I A reference substance 10mg, puts in the brown measuring bottle of 50ml, adds methanol to scale, shakes up; Precision is measured 2ml, puts in the brown measuring bottle of 25ml, adds methanol to scale, shakes up, and promptly gets the reference substance solution that contains tanshinone 16 μ g among every 1ml; Get this product content 0.7g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, put cold, close plug, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains tanshinone must not be less than 0.6mg;
The content assaying method of salvianolic acid B is:
According to an appendix VID of Chinese Pharmacopoeia version in 2010 high performance liquid chromatography, be filler with octadecylsilane chemically bonded silica; With methanol: acetonitrile: formic acid: water=30: 10: 1: 59 is mobile phase; The detection wavelength is 286nm; Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000; It is an amount of that precision takes by weighing the salvianolic acid B reference substance, adds 75% methanol and make the solution that every 1ml contains 0.14mg, promptly gets reference substance solution; Get this product content 0.6g, porphyrize, the accurate title, decide, put in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, through power 120W, frequency 59KHZ supersound process 20 minutes, take out, put cold, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly get need testing solution; Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
Every of this product contains salvianolic acid B must not be less than 6mg.
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| CN102727790A (en) * | 2011-04-08 | 2012-10-17 | 天津同仁堂集团股份有限公司 | Quality control method of nephritis recovery tablets |
| WO2013044567A1 (en) * | 2011-09-30 | 2013-04-04 | 西安千禾药业有限责任公司 | Detection method of drugs for treatment of epileptic seizures, infantile convulsion and facial spasm |
| CN103105443A (en) * | 2013-01-25 | 2013-05-15 | 成都力思特药物研究有限公司 | Method for detecting tanshinone IIA content in yellow spot expelling and freckle removing capsule |
| CN104435108A (en) * | 2014-12-24 | 2015-03-25 | 四川逢春制药有限公司 | Extraction method and detection method of active ingredients of salviae miltiorrhizae stems and leaves |
| CN110716002A (en) * | 2019-06-18 | 2020-01-21 | 南宁市妇幼保健院 | Quality control method of shiwei ginseng-angelica enema liquid |
| CN110646542A (en) * | 2019-09-30 | 2020-01-03 | 贵州中医药大学 | Quality detection method for salvia miltiorrhiza medicinal material |
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