CN101700306A - Quality control method of Rupixiao preparation - Google Patents
Quality control method of Rupixiao preparation Download PDFInfo
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- CN101700306A CN101700306A CN200910213331A CN200910213331A CN101700306A CN 101700306 A CN101700306 A CN 101700306A CN 200910213331 A CN200910213331 A CN 200910213331A CN 200910213331 A CN200910213331 A CN 200910213331A CN 101700306 A CN101700306 A CN 101700306A
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Abstract
The invention relates to a quality control method of Rupixiao preparation; by adopting thin layer chromatography, pseudo-ginseng, radix scrophulariae and red paeony root are distinguished, and content measurement thereof is carried out with high efficiency liquid chromatography by taking ginsenoside Rg1, ginsenoside Rb2 and panax notoginseng saponin as index. All distinguishing methods and all content measurement methods in the quality control method can be used independently or in a combining way or in a cross-use way. The quality control method in the invention has effective quality control of the product, has high precision degree, sensitivity and stability and can ensure the quality of the product to be uniform, stable, effective and controllable.
Description
Technical field
The present invention relates to a kind of method of quality control of Chinese medicine preparation, particularly the method for quality control of Rupixiao preparation.
Background technology
The quality control of Chinese medicine preparation is the key in the preparation process.The Rupixiao preparation crude drug is made up of Cornu Cervi, Herba Taraxaci, Thallus Laminariae (Thallus Eckloniae), Radix Trichosanthis, Caulis Spatholobi, Radix Notoginseng, Radix Paeoniae Rubra, Sargassum, Radix Rhapontici, the Radix Aucklandiae, Radix Scrophulariae, Cortex Moutan, Spica Prunellae, Fructus Forsythiae, Flos Carthami, and it can be made into various dosage forms such as granule, tablet, capsule.In the production practices, need to use the modern medicines analytical technology, the intermediate products and/or the finished product quality of all kinds of dosage forms of RUPIXIAO detected, to guarantee " homogeneous, stable, effective, controlled " of finished product quality.
Summary of the invention
The present invention wants the technical solution problem to be: overcomes the deficiencies in the prior art, a kind of method of quality control of Rupixiao preparation is provided,
The method of quality control that the present invention relates to, comprise detection to raw material, semi-finished product and/or finished product, comprise and differentiate and/or assay that specifically, method of quality control of the present invention comprises with thin layer chromatography to be differentiated Radix Notoginseng, Radix Scrophulariae, Radix Paeoniae Rubra in the Rupixiao preparation; With high performance liquid chromatography with ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1 as index, Rupixiao preparation is carried out assay.
In order to solve above technical problem, the method for quality control of a kind of Rupixiao preparation of the present invention is characterized in that discrimination method in this method of quality control is one or more couplings in the following method:
A. get the about 10g of Rupixiao preparation fine powder, porphyrize adds methanol 50ml, reflux, extract, 40 minutes filters the filtrate evaporate to dryness, residue adds water 20ml makes its dissolving, is transferred in the separatory funnel, uses water saturation n-butanol extraction 3 times, merge extractive liquid,, with the washing of 30ml ammonia solution, discard the ammonia layer, reclaim n-butyl alcohol, residue adds methanol 1ml makes its dissolving, as need testing solution; Other gets Radix Notoginseng control medicinal material 1g, shines medical material solution in pairs with legal system; It is an amount of to get ginsenoside Rg1's reference substance again, adds methanol and makes the solution that every 1ml contains the 0.5mg ginsenoside Rg1, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned three kinds of solution 2～6ul, respectively the cross point is on same silica gel g thin-layer plate, with chloroform-ethyl acetate--and methanol-water is with 10-20: 35-45: 18-26: the 5-15 volume ratio leaves standstill liquid layering to be mixed after mixing, taking off layer solution is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle;
B. get Rupixiao preparation 50g, porphyrize adds methanol 80ml reflux, extract, 40 minutes, filter, solution evaporate to dryness, residue add water 20ml makes its dissolving, be transferred in the separatory funnel, with water saturation n-butanol extraction 3 times, merge extractive liquid,, with the washing of 30ml ammonia solution, discard the ammonia layer, reclaim n-butyl alcohol, residue adds methanol 1ml makes its dissolving, as need testing solution; Other gets Radix Scrophulariae control medicinal material 4g, shines medical material solution in pairs with legal system; According to thin layer chromatography test, draw each 2～6 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 2-8: the 1-4 chloroform-methanol is developing solvent, launches, and takes out, and dries, and spray is with 5% vanillin sulfuric acid solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle;
C. get Rupixiao preparation 50g, porphyrize adds methanol 80ml reflux, extract, 40 minutes, filter, solution evaporate to dryness, residue add water 20ml makes its dissolving, be transferred in the separatory funnel, with water saturation n-butanol extraction 3 times, merge extractive liquid,, with the washing of 30ml ammonia solution, discard the ammonia layer, reclaim n-butyl alcohol, residue adds methanol 1ml makes dissolving, as need testing solution; It is an amount of that other gets the paeoniflorin reference substance, adds methanol and make the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography, draw each 2～6 μ l of need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with 35-45: 1-8: 5-15: 0.1-0.5 chloroform-ethyl acetate-methanol-formic acid is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle.
The method of quality control of above-mentioned Rupixiao preparation is characterized in that:
The developing solvent of method a. is chloroform-ethyl acetate--methanol-water was with 15: 40: 22: 10 volume ratio is mixed, and leaves standstill in 0-20 ℃, after the mixed liquor layering, gets subnatant and carries out chromatography and get;
The developing solvent of method b. is that chloroform-methanol mixes with 5: 1 volume ratios;
The developing solvent of method c. be chloroform-ethyl acetate-methanol-formic acid with 40: 5: 10: 0.2 volume mixture.
The method that the present invention utilizes thin layer chromatography that Radix Notoginseng, Radix Scrophulariae, Radix Paeoniae Rubra in the Rupixiao preparation are differentiated is as follows:
The method of quality control of Rupixiao preparation of the present invention is characterized in that the content assaying method in this method is a high performance liquid chromatography, and content assaying method is as follows:
Chromatographic column is a filler with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A, is Mobile phase B with water;
Gradient elution: 0～12 minute, 19%A, 81%B, 12～60 minutes, 19 → 36%A, 81 → 64%B; The flow velocity per minute is 1.0ml; The detection wavelength is 203nm, and number of theoretical plate calculates by the arasaponin R1 peak should be not less than 20000;
The preparation of reference substance solution: get ginsenoside Rg1's reference substance, ginsenoside Rb1's reference substance and arasaponin R1 reference substance are an amount of, the accurate title, decide, and adds methanol and make the mixed solution that every 1ml contains ginsenoside Rg1 0.4mg, ginsenoside Rb1 0.2mg, arasaponin R1 0.1mg, promptly;
The preparation of need testing solution: the Rupixiao preparation porphyrize, get 10g, the accurate title, decide, put in the tool plug conical flask accurate methanol 50ml, the close plug of adding, claim to decide weight, supersound process 30 minutes is put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with methanol, filter, abandon or adopt filtrate just, precision is measured subsequent filtrate 25ml, put evaporate to dryness in the evaporating dish, residue adds water 15ml makes dissolving, uses water saturation n-butanol extraction 3 times, each 20ml merges n-butyl alcohol liquid, with ammonia solution 30ml thorough washing, discard ammonia solution, the water 30ml washing that the reuse n-butyl alcohol is saturated discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds dissolve with methanol and is transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, get subsequent filtrate, promptly;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
In the unit dose of Rupixiao preparation, contain the total amount of Radix Notoginseng, must not be less than 5.0mg in ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1.
Rupixiao preparation is the RUPIXIAO granule, and every bag of RUPIXIAO granule contains the total amount of Radix Notoginseng in ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1, must not be less than 5.0mg.
Various discrimination methods in the method for quality control of the present invention and various content assaying method both can use respectively separately, also can unite to use or intersect and use.Method of quality control of the present invention is more effective to the quality control of product, and method precision, sensitivity, stability are all higher, can guarantee " homogeneous, stable, effective, controlled " of product quality.
The specific embodiment
The used preparation of each experimental example test sample of the present invention all can adopt embodiment 1 described RUPIXIAO granular preparation, and also available have other preparations that same materials is formed with embodiment 1 described RUPIXIAO granule.
Embodiment 1, RUPIXIAO granule
[prescription] Cornu Cervi 66.8g Herba Taraxaci 44.5g
Thallus Laminariae (Thallus Eckloniae) 173.5g Radix Trichosanthis 17.8g
Caulis Spatholobi 44.5g Radix Notoginseng 44.5g
Radix Paeoniae Rubra 13.4g Sargassum 86.8g
Radix Rhapontici 26.7g Radix Aucklandiae 35.6g
Radix Scrophulariae 44.5g Cortex Moutan 62.3g
Spica Prunellae 44.5g Fructus Forsythiae 17.8g
Flos Carthami 26.7g
[method for making] above 15 flavors, Cornu Cervi, Radix Notoginseng, Radix Scrophulariae powder are broken into impalpable powder, and 12 flavors such as all the other Herba Taraxacis decoct with water 2 times, add for the first time 8 times of decoctings 4 hours, add 6 times of decoctings 3 hours, collecting decoction for the second time, filter, filtrate is concentrated into the clear paste that relative density is 1.30～1.35 (50 ℃).With sucrose 612g and an amount of dextrin, make granule, be drying to obtain.
[discriminating]
(1) get this product and put the microscopically observation: the stone cell yellowish-brown, rectangle like, class garden shape or out-of-shape, laminated striation is obvious, and diameter is translucent to 94um irregular block sheet approximately, and the edge refractive power is stronger, and there are very thin texture and aperture and checking crack in the surface.
(2) get this product 10g, porphyrize adds methanol 50ml, and reflux, extract, 40 minutes filters.Filtrate evaporate to dryness, residue add water 20ml makes dissolving, shifts to put in the separatory funnel, with water saturated n-butyl alcohol jolting extraction 3 times (30,20,20ml), merge extractive liquid, with ammonia solution 30ml washing, discards the ammonia layer, reclaim n-butyl alcohol, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Radix Notoginseng control medicinal material 1g, shines medical material solution in pairs with legal system.It is an amount of to get ginsenoside Rg1's reference substance again, adds methanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005).Draw above-mentioned three kinds of solution, 4 μ l, put respectively on same silica gel g thin-layer plate, (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developing solvent with chloroform-ethyl acetate-methanol-water, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to speckle colour developing at 105 ℃.In the test sample chromatograph, with control medicinal material, the corresponding position of reference substance chromatograph on, show the speckle of same color respectively.
(3) get this product 50g, porphyrize adds methanol 80ml reflux, extract, 40 minutes, filter, solution evaporate to dryness, residue add water 20ml, make dissolving, be transferred in the separatory funnel, with ammonia solution 30ml washing, discard the ammonia layer with water saturation n-butanol extraction 3 times (30,30,20ml) merge extractive liquid,, reclaim n-butyl alcohol, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Radix Scrophulariae control medicinal material 4g, shines material solution in pairs with legal system.Test according to thin layer chromatography S.O.P. (Chinese Pharmacopoeia appendix in 2005), draw above-mentioned two kinds of each 5ul of solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol (5: 1) is developing solvent, launch, taking-up is dried, and spray is with 5% vanillin sulfuric acid solution, and 105 ℃ to be heated to speckle colour developing clear.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle.
(4) get this product 50g, porphyrize adds methanol 80ml reflux, extract, 40 minutes, filter, solution evaporate to dryness, residue add water 20ml, make dissolving, be transferred in the separatory funnel, with ammonia solution 30ml washing, discard the ammonia layer with water saturation n-butanol extraction 3 times (30,30,20ml) merge extractive liquid,, reclaim n-butyl alcohol, residue adds methanol 1ml makes dissolving, as need testing solution.It is an amount of that other gets the paeoniflorin reference substance, adds methanol and make the solution that every 1ml contains 2mg, in contrast product solution.Test according to thin layer chromatography, draw need testing solution and each 4ul of reference substance solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-formic acid (40: 5: 10: 0.2) be developing solvent, launch, taking-up is dried, and spray is with 5% vanillin sulfuric acid solution, and 105 ℃ to be heated to speckle colour developing clear.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle.
[assay] measured according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005).
Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the regulation in the according to the form below is carried out gradient elution; The flow velocity per minute is 1.0ml; The detection wavelength is 203nm.Number of theoretical plate calculates by the arasaponin R1 peak should be not less than 20000.
Ginsenoside Rg1's reference substance is got in the preparation of reference substance solution, ginsenoside Rb1's reference substance and arasaponin R1 reference substance are an amount of, the accurate title, decide, and adds methanol and make the mixed solution that every 1ml contains ginsenoside Rg1 0.4mg, ginsenoside Rb1 0.2mg, arasaponin R1 0.1mg, promptly.
The content under this product content uniformity item is got in the preparation of need testing solution, and porphyrize is got 10g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, close plug claims to decide weight, supersound process (power 250W, frequency 33kHz) 30 minutes, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, abandon or adopt filtrate just, precision is measured subsequent filtrate 25ml, puts evaporate to dryness in the evaporating dish, residue adds water 15ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, each 20ml, merge n-butyl alcohol liquid,, discard ammonia solution with ammonia solution 30ml thorough washing, the water 30ml washing that the reuse n-butyl alcohol is saturated discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds dissolve with methanol and is transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, get subsequent filtrate, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
This product contains the total amount of Radix Notoginseng in ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1, must not be less than 5.0mg.
Embodiment 2, BUPIXIAO PIAN
[prescription] Cornu Cervi 66.8g Herba Taraxaci 44.5g
Thallus Laminariae (Thallus Eckloniae) 173.5g Radix Trichosanthis 17.8g
Caulis Spatholobi 44.5g Radix Notoginseng 44.5g
Radix Paeoniae Rubra 13.4g Sargassum 86.8g
Radix Rhapontici 26.7g Radix Aucklandiae 35.6g
Radix Scrophulariae 44.5g Cortex Moutan 62.3g
Spica Prunellae 44.5g Fructus Forsythiae 17.8g
Flos Carthami 26.7g
[method for making] above 15 flavors, Cornu Cervi, Radix Notoginseng, Radix Scrophulariae powder are broken into impalpable powder, and 12 flavors such as all the other Herba Taraxacis decoct with water 2 times, add for the first time 8 times of decoctings 4 hours, add 6 times of decoctings 3 hours, collecting decoction for the second time, filter, filtrate is concentrated into the clear paste that relative density is 1.30～1.35 (50 ℃).With sucrose 612g and an amount of dextrin, make granule, often regulation becomes tablet, sugar coating or Film coated tablets, 0.34g/ sheet (Film coated tablets) or 0.67g/ sheet (Film coated tablets).
[method of quality control]
Discriminating, content assaying method wherein contain the total amount of Radix Notoginseng in ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1 with embodiment 1, must not be less than 5.0mg.
Embodiment 3, RUPIXIAO capsule
[prescription] Cornu Cervi 66.8g Herba Taraxaci 44.5g
Thallus Laminariae (Thallus Eckloniae) 173.5g Radix Trichosanthis 17.8g
Caulis Spatholobi 44.5g Radix Notoginseng 44.5g
Radix Paeoniae Rubra 13.4g Sargassum 86.8g
Radix Rhapontici 26.7g Radix Aucklandiae 35.6g
Radix Scrophulariae 44.5g Cortex Moutan 62.3g
Spica Prunellae 44.5g Fructus Forsythiae 17.8g
Flos Carthami 26.7g
[method for making] above 15 flavors, Cornu Cervi, Radix Notoginseng, Radix Scrophulariae powder are broken into impalpable powder, and 12 flavors such as all the other Herba Taraxacis decoct with water 2 times, add for the first time 8 times of decoctings 4 hours, add 6 times of decoctings 3 hours, collecting decoction for the second time, filter, filtrate is concentrated into the clear paste that relative density is 1.30～1.35 (50 ℃).With sucrose 612g and an amount of dextrin, make granule, often regulation becomes capsule, the 0.32g/ grain.
[method of quality control]
Discriminating, content assaying method wherein contain the total amount of Radix Notoginseng in ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1 with embodiment 1, must not be less than 5.0mg.
The high performance liquid chromatogram content assaying method research of embodiment 4, ginsenoside and/or arasaponin R1.
(1) instrument, reagent and sample
Instrument: Tianjin, island LC-2010CHT high performance liquid chromatograph, Phenomenex (150 * 4.6mm, 5 μ m), CAPCELL PAK C18 (150 * 4.6mm, 5 μ m) chromatographic column.
Reagent: acetonitrile is a chromatographically pure, and water is purified water, and other reagent is analytical pure.
Reference substance: Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, and for assay usefulness, ginsenoside Rg1's lot number is that 110703-200322, ginsenoside Rb1's lot number are that 110704-200318, arasaponin R1 lot number are 110745-200414.
(2) sample preparation
The preparation of reference substance solution:
Get ginsenoside Rg1's reference substance, ginsenoside Rb1's reference substance and arasaponin R1 reference substance are an amount of, and accurate the title decides, and adds methanol and makes the mixed solution that every 1ml contains ginsenoside Rg1 0.4mg, ginsenoside Rb1 0.2mg, arasaponin R1 0.1mg, promptly.
The preparation of need testing solution:
Get the content under RUPIXIAO granule (embodiment 1) the content uniformity item, porphyrize is got 10g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, close plug claims to decide weight, supersound process (power 250W, frequency 33kHz) 30 minutes, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, abandon or adopt filtrate just, precision is measured subsequent filtrate 25ml, puts evaporate to dryness in the evaporating dish, residue adds water 15ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, each 20ml, merge n-butyl alcohol liquid,, discard ammonia solution with ammonia solution 30ml thorough washing, the water 30ml washing that the reuse n-butyl alcohol is saturated discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds dissolve with methanol and is transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, get subsequent filtrate, promptly.
(3) test condition is selected
1. (whether column temperature important for chromatographic condition? as important, also to list)
Chromatographic column: two pillars have been tried out in front and back: CAPCELL PAK C18 (150 * 4.6mm, 5 μ m) chromatographic column, Phenomenex (150 * 4.6mm, 5 μ m) chromatographic column.As a result, use CAPCELL PAK C18 (150 * 4.6mm, 5 μ m) chromatographic column, the separating degree of ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1 chromatographic peak and other chromatographic peak all can reach more than 1.5; Use Phenomenex (150 * 4.6mm, 5 μ m) chromatographic column, the separating degree of ginsenoside Rg1 and arasaponin R1 chromatographic peak and other chromatographic peak also can reach more than 1.0.Get negative need testing solution sample introduction simultaneously, the result shows that negative test sample does not have respective peaks and occurs in ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1 chromatographic peak position.
2. separating degree should be greater than 1, and number of theoretical plate calculates by the arasaponin R1 peak should be not less than 20000.
(4) linear relationship
It is that 0.4188mg/ml, ginsenoside Rb1's concentration are that 0.187mg/ml, arasaponin R1 concentration are mixing reference substance solution 1 μ l, 4 μ l, 7 μ l, 10 μ l, 13 μ l, 17 μ l, the 20 μ l sample introductions of 0.1002mg/ml that precision is measured ginsenoside Rg1's concentration, the record chromatogram, measure its peak area, the results are shown in Table 1,2,3.And with peak area value (A) to sample size (C) return the standard curve equation.
Table 1 ginsenoside Rg1 reference substance measurement result
Table 2 ginsenoside Rb1 reference substance measurement result
Table 3 arasaponin R1 reference substance measurement result
(5) preparation of need testing solution
1. extract the investigation of solvent: with reference to " content assaying method of a Radix Notoginseng of Chinese pharmacopoeia version in 2005 is chosen methanol for extracting solvent.Once choosing methanol, 50% methanol and three kinds of solvents of ethanol according to bibliographical information investigates ginsenoside Rg1's content, measure its content, the result shows, methanol, two kinds of ginsenoside Rg1's content that solvent extracted of 50% methanol are more or less the same, the content of ethanol extraction is minimum, and with the need testing solution that methanol extracted, impurity peaks is few, separating effect is best, is the extraction solvent of sample so adopt methanol.
2. the investigation of extracting method: precision takes by weighing RUPIXIAO granule 10g, adds methanol 50ml and extracts, and selects three kinds of different extracting method, and promptly soak at room temperature 30 minutes, supersound extraction 30 minutes and reflux, extract, are 30 minutes.The results are shown in Table 4.
The comparison of table 4 Different Extraction Method
| Extracting method | Soak at room temperature 30 minutes | Supersound extraction 30 minutes | Reflux, extract, 30 minutes |
| Arasaponin R1 content (mg/g) | ??0.078 | ??0.086 | ??0.093 |
| Ginsenoside Rg1's content (mg/g) | ??0.332 | ??0.381 | ??0.396 |
| Ginsenoside Rb1's content (mg/g) | ??0.211 | ??0.265 | ??0.253 |
| Total content (mg/g) | ??0.621 | ??0.732 | ??0.743 |
Above result shows that the total content of 30 minutes ginsenoside Rg1 of reflux, extract,, ginsenoside Rb1 and arasaponin R1 is the highest, and the soak at room temperature total content is minimum, is convenient experimental operation, takes all factors into consideration, and determines that extracting method is supersound extraction 30 minutes.
3. the investigation of extraction time: precision takes by weighing RUPIXIAO granule 10g, put in the tool plug conical flask, precision adds methanol 25ml, claims to decide weight, supersound process is 20 minutes, 30 minutes, 40 minutes respectively, put cold back and supply the weight that subtracts mistake with methanol, filter membrane (0.45 μ m) filters, and gets the subsequent filtrate sample introduction, measure its content, supersound process is 30 minutes as a result, and content substantially no longer increases, so determine that extraction time is 30 minutes.
(6) precision experiment
The above-mentioned mixing reference substance solution 10 μ l of accurate absorption repeat sample introduction 5 times, and the RSD of ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1 peak area value shows that less than 3% instrument precision is good as a result, the results are shown in Table 5.
Table 5 Precision test result
| Experiment number | ??1 | ??2 | ??3 | ??4 | ??5 | ??X | ??RSD??(％) |
| The arasaponin R1 peak area | ??280350 | ??279890 | ??280380 | ??284340 | ??284450 | ??281882 | ??0.82 |
| Ginsenoside Rg1's peak area | ??1571200 | ??1561900 | ??1560000 | ??1578600 | ??1578500 | ??1570040 | ??0.56 |
| Ginsenoside Rb1's peak area | ??567170 | ??562780 | ??566760 | ??574670 | ??568210 | ??567918 | ??0.76 |
(7) stability experiment
Getting lot number is that No. 20070401 sample test liquids were respectively at 0 hour, 2 hours, 4 hours, 8 hours, 16 hours sample introduction 10 μ l, the RSD that records ginsenoside Rb1 and arasaponin R1 peak area value in the sample is all less than 3%, the RSD of ginsenoside Rg1's peak area value is less than 4%, test result shows that ginsenoside Rb1 in the test sample, arasaponin R1, ginsenoside Rg1 are basicly stable in 16 hours, the results are shown in Table 6.
Table 6 stability test result
| Time (hour) | ??0 | ??2.5 | ??5 | ??8 | ??16 | ??X | ??RSD??(％) |
| The arasaponin R1 peak area | ??231790 | ??240780 | ??245030 | ??245100 | ??244820 | ??241504 | ??2.37 |
| Ginsenoside Rg1's peak area | ??1423700 | ??1445600 | ??1441800 | ??1424300 | ??1330200 | ??1413120 | ??3.35 |
| Ginsenoside Rb1's peak area | ??807840 | ??771980 | ??786900 | ??776670 | ??773790 | ??783436 | ??1.89 |
(8) repeatability experiment
Press content assaying method, get same lot number sample (lot number is 20070401) and prepare 6 duplicate samples test liquids respectively, record peak area value and calculate content, RSD shows that less than 3% repeatability is good as a result, the results are shown in Table 7.
Table 7 reproducible test results
(9) response rate experiment
Get each 5g of sample (lot number is 20070401) of 6 parts of known content, the accurate title, decide, adding concentration respectively is ginsenoside Rg1's reference substance solution 1.90ml, ginsenoside Rb1's reference substance solution 1.00ml that concentration is 0.935mg/ml, the arasaponin R1 reference substance solution 0.40ml that concentration is 1.002mg/ml of 1.047mg/ml, by sample preparation methods and chromatographic condition in the standard body, the preparation application of sample reclaims need testing solution, and injection high performance liquid chromatograph, with following formula calculate recovery rate, the results are shown in Table 8-10.
Table 8 arasaponin R1 recovery test result
Table 9 ginsenoside Rg1 recovery test result
Table 10 ginsenoside Rb1 recovery test result
(10) test sample assay
Accurate respectively above-mentioned mixing reference substance solution and each 10 μ l of RUPIXIAO particulate samples test liquid of drawing, the chromatographic condition of pressing in the quality standard text is measured, and the results are shown in Table 11.
Assay result (n=2) in table 11 sample
Above data show, arasaponin R1, ginsenoside Rg1 and ginsenoside Rb1's total content is at 5.80 milligrams/bag～6.42 milligrams/bag in the RUPIXIAO granule, 6.09 milligrams/bag of average out to, consider the factor such as source, collecting time, holding conditions and extraction, preparation of medical material, determine that the total content of arasaponin R1, ginsenoside Rg1 and ginsenoside Rb1 in the RUPIXIAO granule must not be less than 5.0mg for every bag.
The present embodiment high performance liquid chromatography as index, is carried out assay to the RUPIXIAO granule with ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1.This determination also is applicable to other Rupixiao preparation.This high-efficient liquid phase technique also is applicable to the assay of raw material, semi-finished product and/or finished product to Rupixiao preparation.
Ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1 are the effective ingredient in the Radix Notoginseng, and therefore, this high-efficient liquid phase technique also can be used for the assay of these three kinds of Saponins in the pharmaceutical composition that Radix Notoginseng or other contain Radix Notoginseng.
This high-efficient liquid phase technique also can be used for other assay that contains the pharmaceutical composition of ginsenoside Rg1, ginsenoside Rb1 and/or arasaponin R1.
The total content of ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1 in embodiment 5, the high effective liquid chromatography for measuring RUPIXIAO granule
Measure with reference to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005).
Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A, is Mobile phase B with water, and the regulation in the according to the form below is carried out gradient elution; The flow velocity per minute is 1.0ml; The detection wavelength is 203nm.Number of theoretical plate calculates by the arasaponin R1 peak should be not less than 20000.
Ginsenoside Rg1's reference substance is got in the preparation of reference substance solution, ginsenoside Rb1's reference substance and arasaponin R1 reference substance are an amount of, the accurate title, decide, and adds methanol and make the mixed solution that every 1ml contains ginsenoside Rg1 0.4mg, ginsenoside Rb1 0.2mg, arasaponin R1 0.1mg, promptly.
The content under this product content uniformity item is got in the preparation of need testing solution, and porphyrize is got 10g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, close plug claims to decide weight, supersound process (power 250W, frequency 33kHz) 30 minutes, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, abandon or adopt filtrate just, precision is measured subsequent filtrate 25ml, puts evaporate to dryness in the evaporating dish, residue adds water 15ml makes dissolving, extracts 3 times with water saturated n-butyl alcohol jolting, each 20ml, merge n-butyl alcohol liquid,, discard ammonia solution with ammonia solution 30ml thorough washing, the water 30ml washing that the reuse n-butyl alcohol is saturated discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds dissolve with methanol and is transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, get subsequent filtrate, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
Every bag of total amount that contains Radix Notoginseng in ginsenoside Rg1 (C42H72014), ginsenoside Rb1 (C54H92023) and arasaponin R1 (C47H80018) of this product must not be less than 5.0mg.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (6)
1. the method for quality control of a Rupixiao preparation is characterized in that discrimination method in this method of quality control is one or more couplings in the following method:
A. get the about 10g of Rupixiao preparation fine powder, porphyrize adds methanol 50ml, reflux, extract, 40 minutes filters the filtrate evaporate to dryness, residue adds water 20ml makes its dissolving, is transferred in the separatory funnel, uses water saturation n-butanol extraction 3 times, merge extractive liquid,, with the washing of 30ml ammonia solution, discard the ammonia layer, reclaim n-butyl alcohol, residue adds methanol 1ml makes its dissolving, as need testing solution; Other gets Radix Notoginseng control medicinal material 1g, shines medical material solution in pairs with legal system; It is an amount of to get ginsenoside Rg1's reference substance again, adds methanol and makes the solution that every 1ml contains the 0.5mg ginsenoside Rg1, in contrast product solution; Test according to thin layer chromatography, draw above-mentioned three kinds of solution, 2～6 μ l, respectively the cross point is on same silica gel g thin-layer plate, with chloroform-ethyl acetate--and methanol-water is with 10-20: 35-45: 18-26: the 5-15 volume ratio leaves standstill liquid layering to be mixed after mixing, taking off layer solution is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle;
B. get Rupixiao preparation 50g, porphyrize adds methanol 80ml reflux, extract, 40 minutes, filter, solution evaporate to dryness, residue add water 20ml makes its dissolving, be transferred in the separatory funnel, with water saturation n-butanol extraction 3 times, merge extractive liquid,, with the washing of 30ml ammonia solution, discard the ammonia layer, reclaim n-butyl alcohol, residue adds methanol 1ml makes its dissolving, as need testing solution; Other gets Radix Scrophulariae control medicinal material 4g, shines medical material solution in pairs with legal system; According to thin layer chromatography test, draw each 2～6 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 2-8: the 1-4 chloroform-methanol is developing solvent, launches, and takes out, and dries, and spray is with 5% vanillin sulfuric acid solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the same color speckle;
C. get Rupixiao preparation 50g, porphyrize adds methanol 80ml reflux, extract, 40 minutes, filter, solution evaporate to dryness, residue add water 20ml makes its dissolving, be transferred in the separatory funnel, with water saturation n-butanol extraction 3 times, merge extractive liquid,, with the washing of 30ml ammonia solution, discard the ammonia layer, reclaim n-butyl alcohol, residue adds methanol 1ml makes dissolving, as need testing solution; It is an amount of that other gets the paeoniflorin reference substance, adds methanol and make the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography, draw each 2～6 μ l of need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with 35-45: 1-8: 5-15: 0.1-0.5 chloroform-ethyl acetate-methanol-formic acid is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and 105 ℃ to be heated to the speckle colour developing clear; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the same color speckle.
2. according to the method for quality control of the described Rupixiao preparation of claim 1, it is characterized in that: the developing solvent of method a. is chloroform-ethyl acetate--methanol-water was with 15: 40: 22: 10 volume ratio is mixed, leave standstill in 0-20 ℃, after the mixed liquor layering, get subnatant and carry out chromatography and get;
The developing solvent of method b. is that chloroform-methanol mixes with 5: 1 volume ratios;
The developing solvent of method c. be chloroform-ethyl acetate-methanol-formic acid with 40: 5: 10: 0.2 volume mixture.
3. the method for quality control of Rupixiao preparation is characterized in that the content assaying method in this method is a high performance liquid chromatography, and content assaying method is as follows:
Chromatographic column is a filler with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A, is Mobile phase B with water;
Gradient elution: 0～12 minute, 19%A, 81%B, 12～60 minutes, 19 → 36%A, 81 → 64%B; The flow velocity per minute is 1.0ml; The detection wavelength is 203nm, and number of theoretical plate calculates by the arasaponin R1 peak should be not less than 20000;
The preparation of reference substance solution: get ginsenoside Rg1's reference substance, ginsenoside Rb1's reference substance and arasaponin R1 reference substance are an amount of, the accurate title, decide, and adds methanol and make the mixed solution that every 1ml contains ginsenoside Rg1 0.4mg, ginsenoside Rb1 0.2mg, arasaponin R1 0.1mg, promptly;
The preparation of need testing solution: the Rupixiao preparation porphyrize, get 10g, the accurate title, decide, put in the tool plug conical flask accurate methanol 50ml, the close plug of adding, claim to decide weight, supersound process 30 minutes is put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with methanol, filter, abandon or adopt filtrate just, precision is measured subsequent filtrate 25ml, put evaporate to dryness in the evaporating dish, residue adds water 15ml makes dissolving, uses water saturation n-butanol extraction 3 times, each 20ml merges n-butyl alcohol liquid, with ammonia solution 30ml thorough washing, discard ammonia solution, the water 30ml washing that the reuse n-butyl alcohol is saturated discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds dissolve with methanol and is transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, get subsequent filtrate, promptly;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
4. the method for quality control of Rupixiao preparation according to claim 3 is characterized in that, in the unit dose of Rupixiao preparation, contains the total amount of Radix Notoginseng in ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1, must not be less than 5.0mg.
5. the method for quality control of Rupixiao preparation according to claim 3, it is characterized in that, Rupixiao preparation is the RUPIXIAO granule, and every bag of RUPIXIAO granule contains the total amount of Radix Notoginseng in ginsenoside Rg1, ginsenoside Rb1 and arasaponin R1, must not be less than 5.0mg.
6. according to the method for quality control of any described Rupixiao preparation of claim 1-4, it is characterized in that described Rupixiao preparation is the RUPIXIAO granule.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103454374A (en) * | 2013-08-29 | 2013-12-18 | 贵州维康药业有限公司 | Quality control method of bone rehabilitation medicine |
| CN105548425A (en) * | 2016-01-11 | 2016-05-04 | 山东步长制药股份有限公司 | High-performance liquid phase detection method for heart-calming granules |
| CN110702822A (en) * | 2019-02-25 | 2020-01-17 | 广州白云山医药集团股份有限公司白云山何济公制药厂 | Method for detecting content of pseudo-ginseng in pseudo-ginseng traumatic rheumatism ointment |
| CN117169386A (en) * | 2023-09-26 | 2023-12-05 | 广东省科学院生物与医学工程研究所 | Method for detecting content of laminaria oligosaccharide in health-care product |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103454374A (en) * | 2013-08-29 | 2013-12-18 | 贵州维康药业有限公司 | Quality control method of bone rehabilitation medicine |
| CN105548425A (en) * | 2016-01-11 | 2016-05-04 | 山东步长制药股份有限公司 | High-performance liquid phase detection method for heart-calming granules |
| CN110702822A (en) * | 2019-02-25 | 2020-01-17 | 广州白云山医药集团股份有限公司白云山何济公制药厂 | Method for detecting content of pseudo-ginseng in pseudo-ginseng traumatic rheumatism ointment |
| CN110702822B (en) * | 2019-02-25 | 2020-08-04 | 广州白云山医药集团股份有限公司白云山何济公制药厂 | Method for detecting content of pseudo-ginseng in pseudo-ginseng traumatic rheumatism ointment |
| CN117169386A (en) * | 2023-09-26 | 2023-12-05 | 广东省科学院生物与医学工程研究所 | Method for detecting content of laminaria oligosaccharide in health-care product |
| CN117169386B (en) * | 2023-09-26 | 2024-06-11 | 广东省科学院生物与医学工程研究所 | Method for detecting content of laminaria oligosaccharide in health-care product |
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