CN102008683A - Quality detection method for Naokangtai tablets - Google Patents
Quality detection method for Naokangtai tablets Download PDFInfo
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Abstract
The invention discloses a quality detection method for Naokangtai tablets. The method comprises the following steps: (1) identification, wherein petroleum ether (30-60 DEG C)-ether-glacial acetic acid (10:10:0.5) as a developing agent; and (2) content determination, wherein octadecylsilane chemically bonded silica is utilized as a filling agent, methanol-acetonitrile-formic acid-water (30:10:1:59) is utilized as a mobile phase, and the detection wavelength is 286nm. The umber of theoretical plates is not lower than 2000 according to the calculation by salvianolic acid B peak; test solution is prepared; and the determination method is as follows: respectively precisely sucking 10 mu l of control solution and 10 mu l of the test solution, injecting into a liquid chromatograph and carrying out the determination. Each tablet of the product contains not less than 2.0mg of radix salviae miltiorrhizae calculated by salvianolic acid B. The quality detection method for the Naokangtai tablets is safe and high in detection accuracy.
Description
Technical field
The invention belongs to the field of quality control of Chinese medicine preparation, particularly the quality determining method of safe of brain.
Background technology
Safe of brain is by Moschus 0.6g, Rhizoma Chuanxiong 60g, Flos Carthami 90g, Pheretima 90g, Radix Salviae Miltiorrhizae 150g, Rhizoma Curcumae 90g, Semen Persicae 90g, rhizoma sparganic 90g.More than eight the flavor, Flos Carthami, Pheretima powder are broken into fine powder, and be standby; All the other five tastes except that Moschus add 8 times, 6 times water gagings respectively and decoct secondary, each 1.5 hours, collecting decoction, it is the clear paste of 1.10～1.15 (70 ℃) that concentrating under reduced pressure becomes relative density, carries out boiling granulating, drying with above-mentioned fine powder and microcrystalline Cellulose 92.4g, granulate (crossing 20 mesh sieves), standby; Other gets Moschus and mixes with the 15.6g microcrystalline Cellulose and pulverized 80 mesh sieves, mixes with granule, magnesium stearate 2g again, is pressed into 1000, the bag film-coat, promptly.This product is a Film coated tablets, removes to show yellowish-brown behind the coating to sepia; Gas perfume (or spice), mildly bitter flavor.Function cures mainly: blood circulation promoting and blood stasis dispelling.Be used for apoplexy, apoplex involving the channels and collaterals belongs to syndrome of static blood blocking collaterals, card is seen: hemiplegia, language is puckery not smoothgoing.
Safe of brain is to be got by medicine " the safe capsule of the brain " dosage changing form of existing national standard.It is developing solvent that the safe capsule quality standard of protocerebrum archicerebrum is differentiated in (2) with benzene-ethyl acetate-formic acid (4: 1: 0.1), and wherein benzene is bigger to the human body murder by poisoning.Assay in the former dosage form standard is that the contained Tanshinone I I A. of red rooted salvia shows that according to data effective component in red sage is the diterpene compound (Tanshinone I, Tanshinone I I A, Tanshinone I I B etc.) and the water miscible phenolic acid compound (danshensu, salvianolic acid B etc.) of ester dissolubility in the detection prescription.What wherein cardiovascular system is worked mainly is the water soluble ingredient of Radix Salviae Miltiorrhizae, detect Tanshinone I I A content difficulty and reach primary standard content limit requirement (former limit is the 0.03mg/ grain), be lower than ten thousand/, it is inappropriate that visible former dosage form selection Tanshinone I I A makes the assay index.
Summary of the invention
The quality determining method that the high brain of a kind of safety, the detection accuracy that the objective of the invention is to overcome above-mentioned shortcoming and provide is safe.
The quality determining method that a kind of brain of the present invention is safe comprises the steps:
(1) differentiate:
A, get 10 of this product, remove coating, porphyrize, put microscopically and observe: the irregular shape agglomerate that unsetting granular substance is integrated, faint yellow or light brown is embedded with square, cylindricality or irregular shape crystal in the agglomerate; The pollen grain similar round, the about 60 μ m of diameter, some has 3 germinal aperatures, and outer wall has dentation.Colourless muscle fiber or light brown are arranged, loose from or mutually according to knot, crooked or straight mostly;
B, get 20 of this product, remove coating, porphyrize is got about 5g, the 50ml that adds diethyl ether, and supersound process (power 250W, frequency 34KHZ) 1 hour is put coldly, filters, and filtrate is put evaporate to dryness in the water-bath, and residue adds ethyl acetate 2ml makes dissolving, as need testing solution.Other gets the ferulic acid reference substance, adds ethyl acetate and makes the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with petroleum ether (30～60 ℃)-ether-glacial acetic acid (10: 10: 0.5) is developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
C, get 20 of this product, remove coating, porphyrize is got about 3g, adds 80% acetone 10ml, and jolting 15 minutes filters, and gets subsequent filtrate, as need testing solution.Other gets Flos Carthami control medicinal material 0.5g, gets control medicinal material solution with legal system.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-formic acid-water-methanol (7: 2: 3: 0.4) be developing solvent, launch, take out, dry.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
(2) assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
A, chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-acetonitrile-formic acid-water (30: 10: 1: 59) be mobile phase; The detection wavelength is 286nm.Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000;
The preparation of B, reference substance solution: it is an amount of to get the salvianolic acid B reference substance, and accurate the title decides, and adds 75% methanol and makes the solution that every 1ml contains 35 μ g, promptly;
The preparation of C, need testing solution: get 20 of this product, remove coating, porphyrize is got about 0.2g, and accurate the title decides, and puts in the tool plug conical flask, and the accurate 75% methanol 50ml that adds claims to decide weight, supersound process (power 250W, frequency 34KH
Z) 1 hour, put coldly, claim again decide weight, supply the weight that subtracts mistake with 75% methanol, shake up, filtration is got subsequent filtrate, promptly;
D, algoscopy: 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 Radix Salviae Miltiorrhizae with salvianolic acid B (C
36H
30O
16) meter, must not be less than 2.0mg.
The present invention compared with prior art has tangible beneficial effect, by above technical scheme as can be known, is developing solvent with petroleum ether (30～60 ℃)-ether-glacial acetic acid (10: 10: 0.5) in the discriminating, and is harmless, guaranteed safety.Select the water soluble ingredient-salvianolic acid B of the then quite high and Radix Salviae Miltiorrhizae that cardiovascular system is worked of content for use, just in time cure mainly and be consistent with the method for making of this product and function.Therefore this product will contain and survey index components and be defined as surveying salvianolic acid B.Thereby guaranteed the accuracy of testing result.Guarantee the safety of medication, better instruct and produce, make technology controlling and process rationally strict more.
The specific embodiment
Below by testing the beneficial effect that example further specifies the quality determining method of safe of brain of the present invention:
The test example:
(1) differentiates
1, instrument, reference substance, control medicinal material, reagent and reagent:
Instrument: the automatic bed board device of BYCDE thin layer; Electronic balance (ten thousand/) BS-210S type.
Reagent: ethyl acetate, formic acid, acetone, petroleum ether (60～90 ℃), ether, glacial acetic acid, cyclohexane extraction are analytical pure.
Reference substance: ferulic acid reference substance (lot number: 0773-9910) provide by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Control medicinal material: flos carthami, rhizoma sparganic medical material, Pheretima medical material, red rooted salvia through accreditation medical material use in contrast, are provided by Guiyang Xintian Pharmaceutical Industry Co., Ltd..
Reagent: safe of brain is provided by Guiyang Xintian Pharmaceutical Industry Co., Ltd..
2, differentiate (1): this discriminatings is microscopical identification " an appendix II of Chinese pharmacopoeia C medical material and prescribed preparation microscopical identification method are differentiated with reference to the safe capsule of brain and 2005 editions.
Get 20 of this product, remove coating, porphyrize, put microscopically and observe: the irregular shape agglomerate that unsetting granular substance is integrated, yellow or light brown are embedded with square, cylindricality or irregular shape crystal in the agglomerate; The pollen grain similar round, the about 60 μ m of diameter, some has 3 germinal aperatures, and outer wall has dentation.Colourless or the light brown of muscle fiber, loose from or mutually according to knot, crooked or straight mostly.
Conclusion: this product microscopic features are obvious, so list it in text.
3, differentiate (2): this discriminating is differentiated for the Rhizoma Chuanxiong thin layer.
3.1 the selection of extraction conditions is differentiated (2) with reference to the safe capsule quality standard of brain.
The safe capsule quality standard of brain differentiates that sample treatment is in (2): get content 5g, add water 30ml, reflux 2 hours, put cold, filter, filtrate adds dilute hydrochloric acid and regulates pH value to 2, and the jolting of reuse ethyl acetate is extracted 2 times, each 20ml, combined ethyl acetate liquid is used anhydrous sodium sulfate dehydration, evaporate to dryness, residue adds ethyl acetate 1ml makes dissolving, promptly.This operation is too complicated, therefore extraction conditions is changed into: get 20 of this product, remove coating, porphyrize, get about 5g, the 50ml that adds diethyl ether, supersound process (power 250W, frequency 34KHZ) 1 hour, put coldly, filter, filtrate is put evaporate to dryness in the water-bath, residue adds ethyl acetate 2ml makes dissolving, as need testing solution; It is an amount of that other gets the ferulic acid reference substance, adds ethyl acetate and make the reference substance solution that contains 1mg among every 1ml.Through overtesting, this method separating effect is better, so list text in.
3.2 the selection of adsorbent is differentiated (2) with reference to the safe capsule quality standard of brain, selecting silica gel G for use is that the adsorbent separating effect is better, so list it in text.
3.3 it is developing solvent that the safe capsule quality standard of the selection brain of developing solvent is differentiated in (2) with benzene-ethyl acetate-formic acid (4: 1: 0.1), wherein benzene is poisoned bigger to human body, so it is changed into: with petroleum ether (30～60 ℃)-ether-glacial acetic acid (10: 10: 0.5) is developing solvent, launch, take out, dry, under uviol lamp (365nm), inspect.Good separating effect between each speckle as a result, and negative control is noiseless, therefore lists this developing solvent in text.
In sum, determined text Rhizoma Chuanxiong TLC inspection method, through 3 batches of pilot scale sampling tests, this method is stable, favorable reproducibility, and negative noiseless.
4, differentiate (3): the thin layer of Flos Carthami is differentiated in this discriminating side of being.
4.1 the selection of extraction conditions
Get 20 of this product, remove coating, porphyrize is got about 3g, adds 80% acetone 10ml, and jolting 15 minutes filters, and gets subsequent filtrate, as need testing solution.Other gets flos carthami 0.5g, gets control medicinal material solution with legal system.Through overtesting, this method separating effect is better, so list text in.
Separate 4.2 the selection of adsorbent employing sodium carboxymethyl cellulose is the silica gel H plate of adhesive, effect is better, so list it in text.
(7: 2: 3: 0.4) be developing solvent, expansion was taken out, and dries 4.3 the selection of developing solvent is with ethyl acetate-formic acid-water-methanol.Good separating effect between each speckle as a result, and negative control is noiseless, therefore lists this developing solvent in text.
In sum, determined the Flos Carthami TLC inspection method of text, through 3 batches of pilot scale sampling tests, this method is stable, favorable reproducibility, and negative noiseless.
In addition, the inventor goes back rhizoma sparganic, Pheretima among the other side, has carried out the TLC discriminating, and method is infeasible as a result, so exclude quality standard draft text.
(2) check
1, general inspection
1.1 content uniformity is by the following regulation of (appendix ID of Chinese Pharmacopoeia version in 2005) tablet item, limit test of weight variation should heavy at designation card (or average sheet amount) ± 5% in, what exceed the content uniformity limit must not be more than 2, and 1 times of 1 overrun must not be arranged.Check that in accordance with the law the result is all up to specification.
1.2 disintegration, each sheet should all disintegrates in 1 hour by (an appendix XII of Chinese Pharmacopoeia version in 2005 A) inspection technique disintegration regulation.Check that in accordance with the law the result is all up to specification.
1.3 limit test of microbe is by " " microbial limit standard " regulation of Chinese pharmacopoeia version in 2005, tablet bacterial population per 1 must not restrain 10000, mycete, yeast count per 1 must not restrain above 100, escherichia coli per 1 must not restrain and detected, per 1 gram of coliform is less than 100, and the demodicid mite that lives must not detect.This preparation testing result is all up to specification.
2, heavy metal and arsenic salt are checked
2.1 heavy metal inspection: by " 2005 editions one appendix IX E of Chinese pharmacopoeia heavy metal inspection technique, the second method inspection.The result is less than 10/1000000ths.
2.2 arsenic salt is checked: by " 2005 editions one appendix IX F of Chinese pharmacopoeia arsenic salt inspection technique, the first method inspection.The result is less than 2/1000000ths.
According to above assay, illustrate that heavy metal in this product, arsenic salt are all up to specification, and content is all very low, therefore do not list text in.
(3) assay is measured according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 D).
Assay in the former dosage form standard of this product is that the contained Tanshinone I I A. of red rooted salvia shows that according to data effective component in red sage is the diterpene compound (Tanshinone I, Tanshinone I I A, Tanshinone I I B etc.) and the water miscible phenolic acid compound (danshensu, salvianolic acid B etc.) of ester dissolubility in the detection prescription.What wherein cardiovascular system is worked mainly is the water soluble ingredient of Radix Salviae Miltiorrhizae, and this cures mainly with the method for making of this product and function and is consistent.We are under the qualified situation of red rooted salvia Tanshinone I I A content, manufacture experimently by the safe capsule technology of brain, sample detects Tanshinone I I A and salvianolic acid B respectively, the former content does not reach primary standard content limit requirement (former limit is the 0.03mg/ grain) as a result, be lower than ten thousand/, latter's content is then quite high.We think that it is inappropriate that former dosage form selection Tanshinone I I A makes the assay index, so this product will contain and survey index components and change into and survey salvianolic acid B (testing result sees Table 1)
Table 1
1, instrument, reference substance, reagent and reagent
1.1 instrument: Agilent 1100 type high performance liquid chromatographs, VWD detector, Agilent chem workstation;
Ultrasonic cleaner CX-250 type (frequency: 29-34KHz, power: 〉=250W, Beijing armarium two factories);
Electronic balance (ten thousand/) BS-210S type (Sai Duolisi company); Electronic balance (100,000/) AE240 type (Mettler company).
1.2 reference substance: salvianolic acid B reference substance (lot number: 111562-200403), purchase in Nat'l Pharmaceutical ﹠ Biological Products Control Institute for assay usefulness.
1.3 reagent: formic acid is analytical pure; Methanol, acetonitrile are chromatographically pure; Water is double distilled water.
1.4 reagent: safe sample of brain provided by Guiyang Xintian Pharmaceutical Industry Co., Ltd..
2, the preparation of reference substance solution
Reference substance solution 1. precision takes by weighing salvianolic acid B reference substance 14.0mg, puts in the 100ml measuring bottle, adds 75% dissolve with methanol and is diluted to scale, shakes up, and promptly gets the solution that every 1ml contains 140.0 μ g.
Reference substance solution 2. precision is measured 1. 5ml of reference substance solution, puts in the 20ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets the solution that every 1ml contains 35.0 μ g.
3, chromatographic condition test
Chromatographic column: octadecylsilane chemically bonded silica is filler (Diamonsil (TM) diamond C18 250mm * 4.6mm, 5 μ m)
Flow velocity: 1.0ml/ minute
Detect wavelength: 286nm
3.1 the selection of mobile phase:
Get 20 of this product, remove coating, porphyrize, get about 0.2g, 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 250W, frequency 34KHZ) 1 hour, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, as need testing solution.
(30: 10: 1: 59) be mobile phase, accurate respectively reference substance solution (C=35.0 μ g/ml) and each 10 μ l of need testing solution of drawing injected chromatograph of liquid, the record chromatogram with methanol-acetonitrile-formic acid-water.(collection of illustrative plates is seen the safe chip system compatibility test of brain collection of illustrative plates).
The result as can be seen, has good separating between salvianolic acid B peak and other peak from the test sample collection of illustrative plates, and peak shape is good, post is imitated highly, shows that this mobile phase and related color spectral condition can be used as the chromatographic condition of this experiment.
4, the preparation of need testing solution is selected:
Method 1: get 20 of this product, remove coating, porphyrize, get about 0.2g, 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 250W, frequency 34KHZ) 1 hour, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly.
Method 2: get 20 of this product, remove coating, porphyrize, get about 0.2g, 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 250W, frequency 34KHZ) 30 minutes, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly.
Method 3: get 20 of this product, remove coating, porphyrize, get about 0.2g, 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 250W, frequency 34KHZ) 90 minutes, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly.
Method 4: get 20 of this product, remove coating, porphyrize is got about 0.2g, and accurate the title decides, and puts in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims decide weight, and heating and refluxing extraction 1 hour is put coldly, and weight decided in title again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly.
Respectively accurate 1. 2. (140.0 μ g/ml) and 4 each the 10 μ l of need testing solution of (C=35.0 μ g/ml), salvianolic acid B reference substance solution of salvianolic acid B reference substance solution that draw inject chromatograph of liquid, write down chromatogram, the results are shown in Table 2.
Table 2
Conclusion: from above result of the test as can be seen, in each method, between salvianolic acid B peak and other peak good separating arranged all, method 2,4 content are low, and method 1,3 content do not have significant change, take all factors into consideration so list method 1 in text, be: get 20 of this product, remove coating, porphyrize, get about 0.2g, 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 250W, frequency 34KHZ) 1 hour is put cold, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, promptly.
5, system suitability test
Get 20 of this product, remove coating, porphyrize, get about 0.2g, 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 250W, frequency 34KHZ) 1 hour, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, as need testing solution; Other gets negative sample and makes negative sample solution with method.Accurate each the 10 μ l of reference substance solution (35.0 μ g/ml), negative sample solution and need testing solution that draw inject chromatograph of liquid, the record chromatogram.From chromatogram, can find out, test sample with the corresponding position of reference substance retention time on the peak is arranged, and peak shape is good, and negative sample does not have the peak on corresponding position, illustrate that negative sample is noiseless to sample determination.The number of theoretical plate at the salvianolic acid B peak of sample in collection of illustrative plates is 4394, with other material good separating is arranged, and peak shape is good, takes all factors into consideration, and stipulates that the theoretical cam curve of this experiment is calculated by the salvianolic acid B peak, should be lower than 2000.
6, linear relationship test
The reference substance stock solution: precision takes by weighing salvianolic acid B reference substance 14.00mg and puts in the 100ml measuring bottle, adds 75% dissolve with methanol and is diluted to scale, shakes up, and promptly gets and whenever promptly gets the solution that every 1ml contains 0.14mg.
Reference substance solution is 1.: the accurate reference substance stock solution 5ml that draws, put in the 20ml measuring bottle, and add 75% dissolve with methanol and be diluted to scale, shake up, the accurate again 5ml that draws puts in the 20ml measuring bottle, add 75% dissolve with methanol and be diluted to scale, shake up, promptly get the solution that every 1ml contains 0.00875mg.
Reference substance solution is 2.: the accurate 1. reference substance solution 1ml after the dilution for the first time of above-mentioned reference substance solution that draws, put in the 2ml measuring bottle, and add 75% dissolve with methanol and be diluted to scale, shake up, promptly get the solution that every 1ml contains 0.0175mg.
Reference substance solution is 3.: precision takes by weighing salvianolic acid B reference substance 14.90mg, puts in the 50ml measuring bottle, adds 75% dissolve with methanol and is diluted to scale, shake up, the accurate again 1ml that draws puts in the 10ml measuring bottle, add 75% methanol and be diluted to scale, shake up, promptly get the solution that every 1ml contains 0.0298mg.
Reference substance solution is 4.: the accurate 3. reference substance solution 5ml after the dilution for the first time of above-mentioned reference substance solution that draws, put in the 20ml measuring bottle, and add 75% dissolve with methanol and be diluted to scale, shake up, promptly get the solution that every 1ml contains 0.0745mg.
Reference substance solution is 5.: precision takes by weighing salvianolic acid B reference substance 12.08mg, puts in the 100ml measuring bottle, adds 75% dissolve with methanol and is diluted to scale, shakes up, and promptly gets the solution that every 1ml contains 0.1208mg.
Accurate each the 10 μ l of above-mentioned 5 reference substance solution that draw inject chromatograph of liquid, record chromatogram, table 3 as a result.
Table 3
| Sequence number | 1 | 2 | 3 | 4 | 5 |
| Reference substance solution concentration (mg/ml) | 0.00875 | 0.0175 | 0.0298 | 0.0745 | 0.1208 |
| Peak area | 101.26515 | 207.56990 | 360.21643 | 834.85413 | 1421.82837 |
With the peak area is vertical coordinate, is that abscissa is made linear relationship chart with concentration (mg/ml)
Get linear equation Y=11634x+0.293 R=0.9994
The linear equation that initial point is crossed in match is: Y=11638x R=0.9994
Above-mentioned two formulas of reference substance peak area substitution are calculated, and relative standard deviation is less than 1.0%, so can think that standard curve crosses initial point, the regression equation intercept is zero, and assay can adopt one point external standard method calculating thus.Reference substance solution concentration linear relationship between 0.00875mg/ml～0.1208mg/ml is good.
7, accurate salvianolic acid B reference substance solution (C=35.0 μ g/ml) the 10 μ l that draw of precision test inject chromatograph of liquid, continuous sample introduction 5 times, and the record chromatogram the results are shown in Table 4:
Table 4
Experimental result shows that this method has good precision.
8, sample stability test:
Get 20 of this product, remove coating, porphyrize, get about 0.2g, 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 250W, frequency 34KHZ) 1 hour, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, as need testing solution.The respectively accurate need testing solution 10 μ l that draw inject chromatograph of liquid, and record chromatogram, and after need testing solution placed 0,2,4,12 hour is accurately respectively drawn need testing solution 10 μ l sample introductions and measured, and the record chromatogram the results are shown in Table 5:
Table 5
Draw from above-mentioned result of the test, need testing solution is good at 12 hours internal stabilities.
9, the heavy property of sample test
Get 20 of this product, remove coating, porphyrize, get about 0.2g, the accurate title, decided (5 parts in parallel sample), puts in the tool plug conical flask, the accurate 75% methanol 50ml that adds claims to decide weight, supersound process (power 250W, frequency 34KHZ) 1 hour, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, as need testing solution.Accurate respectively reference substance solution (concentration is 140.0 μ g/ml) and five parts of each 10 μ l sample introductions mensuration of need testing solution drawn the results are shown in Table 6:
Table 6
Draw the sample favorable reproducibility of this method by above-mentioned result of the test.
10, sample pipetting volume recovery test
Get this product (lot number: 20060201) 20, remove coating, porphyrize, get about 0.1g (6 parts in parallel sample), the accurate title, decide, and puts in the conical flask, accurate salvianolic acid B reference substance 75% methanol solution (C=0.7mg/50ml) 50ml that adds claims to decide weight, supersound process (power 250W, frequency 34KHZ) 1 hour, put coldly, claim to decide weight again, supply the weight that subtracts mistake with 75% methanol, shake up, filter, get subsequent filtrate, as need testing solution.Accurate respectively salvianolic acid B reference substance solution (C=35.0 μ g/ml) and each 10 μ l of above-mentioned 6 parts of need testing solutions of drawing, sample introduction is measured respectively, the results are shown in Table 7:
Table 7
Draw from above-mentioned experimental result, this method has the good response rate.
11, sample determination
Three batches of pilot scale sample size measurement results see Table 8:
Table 8
| Lot number | Content 1 (mg/ sheet) | Content 2 (mg/ sheet) | Average content (mg/ sheet) |
| 20060201 | 3.63 | 3.60 | 3.6 |
| 20060202 | 3.53 | 3.54 | 3.5 |
| 20060203 | 3.59 | 3.57 | 3.6 |
The content limit computing formula:
According to aforementioned calculation formula result of calculation is the 2.06mg/ sheet, so every of regulation this product contains Radix Salviae Miltiorrhizae with salvianolic acid B (C
36H
30O
166) meter must not be less than 2.0mg.
Embodiment:
The quality determining method that a kind of brain is safe comprises the steps:
(1) differentiate:
A, get 10 of this product, remove coating, porphyrize, put microscopically and observe: the irregular shape agglomerate that unsetting granular substance is integrated, faint yellow or light brown is embedded with square, cylindricality or irregular shape crystal in the agglomerate; The pollen grain similar round, the about 60 μ m of diameter, some has 3 germinal aperatures, and outer wall has dentation.Colourless muscle fiber or light brown are arranged, loose from or mutually according to knot, crooked or straight mostly;
B, get 20 of this product, remove coating, porphyrize is got about 5g, the 50ml that adds diethyl ether, and supersound process (power 250W, frequency 34KHZ) 1 hour is put coldly, filters, and filtrate is put evaporate to dryness in the water-bath, and residue adds ethyl acetate 2ml makes dissolving, as need testing solution.Other gets the ferulic acid reference substance, adds ethyl acetate and makes the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with petroleum ether (30～60 ℃)-ether-glacial acetic acid (10: 10: 0.5) is developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
C, get 20 of this product, remove coating, porphyrize is got about 3g, adds 80% acetone 10ml, and jolting 15 minutes filters, and gets subsequent filtrate, as need testing solution.Other gets Flos Carthami control medicinal material 0.5g, gets control medicinal material solution with legal system.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-formic acid-water-methanol (7: 2: 3: 0.4) be developing solvent, launch, take out, dry.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
(2) assay: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
A, chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-acetonitrile-formic acid-water (30: 10: 1: 59) be mobile phase; The detection wavelength is 286nm.Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000;
The preparation of B, reference substance solution: it is an amount of to get the salvianolic acid B reference substance, and accurate the title decides, and adds 75% methanol and makes the solution that every 1ml contains 35 μ g, promptly;
The preparation of C, need testing solution: get 20 of this product, remove coating, porphyrize is got about 0.2g, and accurate the title decides, and puts in the tool plug conical flask, and the accurate 75% methanol 50ml that adds claims to decide weight, supersound process (power 250W, frequency 34KH
Z) 1 hour, put coldly, claim again decide weight, supply the weight that subtracts mistake with 75% methanol, shake up, filtration is got subsequent filtrate, promptly;
D, algoscopy: 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 Radix Salviae Miltiorrhizae with salvianolic acid B (C
36H
30O
16) meter, must not be less than 2.0mg.
Claims (2)
1. the quality determining method of safe an of brain comprises the steps:
(1) differentiate:
A, get 10 of this product, remove coating, porphyrize, put microscopically and observe: the irregular shape agglomerate that unsetting granular substance is integrated, faint yellow or light brown is embedded with square, cylindricality or irregular shape crystal in the agglomerate; The pollen grain similar round, the about 60 μ m of diameter, some has 3 germinal aperatures, and outer wall has dentation; Colourless muscle fiber or light brown are arranged, loose from or mutually according to knot, crooked or straight mostly;
B, get 20 of this product, remove coating, porphyrize is got about 5g, the 50ml that adds diethyl ether, and power 250W, frequency 34KHZ supersound process 1 hour are put coldly, filter, and filtrate is put evaporate to dryness in the water-bath, and residue adds ethyl acetate 2ml makes dissolving, as need testing solution; Other gets the ferulic acid reference substance, adds ethyl acetate and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw each 5～10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 30～60 ℃ of petroleum ether: ether: glacial acetic acid=10: 10: 0.5 is developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
C, get 20 of this product, remove coating, porphyrize is got about 3g, adds 80% acetone 10ml, and jolting 15 minutes filters, and gets subsequent filtrate, as need testing solution; Other gets Flos Carthami control medicinal material 0.5g, gets control medicinal material solution with legal system; According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate: formic acid: water: methanol=7: 2: 3: 0.4 be developing solvent, launches, and taking-up is dried; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(2) assay:
A, chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; 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;
The preparation of B, reference substance solution: it is an amount of to get the salvianolic acid B reference substance, and accurate the title decides, and adds 75% methanol and makes the solution that every 1ml contains 35 μ g, promptly;
The preparation of C, need testing solution: get 20 of this product, remove coating, porphyrize is got about 0.2g, and accurate the title decides, and puts in the tool plug conical flask, and the accurate 75% methanol 50ml that adds claims to decide weight, power 250W, frequency 34KH
ZSupersound process 1 hour is put coldly, claims decide weight again, supplies the weight that subtracts mistake with 75% methanol, shakes up, and filtration is got subsequent filtrate, promptly;
D, algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
2. the quality determining method that brain as claimed in claim 1 is safe, wherein: every of this product contains Radix Salviae Miltiorrhizae in salvianolic acid B, must not be less than 2.0mg.
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| CN115561380A (en) * | 2021-12-29 | 2023-01-03 | 贵州威利德制药有限公司 | Detection method of salvia miltiorrhiza ointment |
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Open date: 20110413 |