CN103063799A - Method for comprehensively detecting active ingredients of Longdan Xiegan particles - Google Patents
Method for comprehensively detecting active ingredients of Longdan Xiegan particles Download PDFInfo
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Abstract
The invention relates to a method for detecting active ingredients of Longdan Xiegan particles and belongs to the field of modernization of Chinese traditional medicine. According to the invention, the method for detecting the Longdan Xiegan particles is standardized; on the basis of original identification on gardenia, by establishment of identification and content determination on gentian, identification and content determination on baical skullcap root and liquorice and content determination on gardenia serving as ministerial drug, ingredients of the medicament and compatibility of the ingredients can be more comprehensively detected and content and stability of the Longdan Xiegan particles can be more comprehensively known; the method is also helpful to explaining the medicinal material basis of the medicament; and meanwhile, due to increase of examination on aristolochic acid, the method is more beneficial for reducing toxic and side effects of the Longdan Xiegan particles.
Description
Technical field
The invention belongs to modern Chinese traditional medicine field, be specifically related to a kind of detection method of Longdanxiegan particle.
Background technology
Traditional Chinese medicine ingredients is complicated, and particularly Chinese medicine compound prescription often contains multiclass, Multiple components, has consisted of a very complicated system.At present, new active component Chinese medicine preparation and the research and development of modern Chinese traditional compound medicine are on the increase, and new technique and multiple different extracting mode also are used for research and the production of medicine.
Along with the fast development of Modern Testing, equipment and instrument, the assay of Chinese medicine, chromatographic identification become the important means of detection.But assay or the discriminating of at present generally only setting up a certain index chemical constitution for the simultaneously applied situation of above-mentioned multiple new technology, are only set up the assay of certain single component or are differentiated to a great extent with larger one-sidedness.
The Longdanxiegan particle has the clearing liver courage, the function of dampness removing heat.Be used for liver and gallbladder damp-heat, dizziness and red eyes, Hiccough and deaf, the ear pain that swells, the hypochondriac pain bitter taste is urinated red puckery pain, under the humid tropics.
The Longdanxiegan particle is yellow to tan particle; The little perfume (or spice) of gas, the flavor sweet, little hardship.
Its prescription is: rough gentian 66g, radix bupleuri 66g, root of large-flowered skullcap 33g, cape jasmine (stir-fry) 33g, rhizoma alismatis 66g, akebi 33g, plantain seed (salt stir-fry) 33g, Radix Angelicae Sinensis (wine stir-fry) 33g, glutinous rehmannia 66g, Radix Glycyrrhizae (honey is processed) 33g.
Method for making: above ten flavors, get radix bupleuri, Radix Angelicae Sinensis extraction volatile oil, the aqueous solution after the distillation in addition device is collected; Eight flavors such as the dregs of a decoction and all the other rough gentian, the boiling secondary adds 8 times of water gagings for the first time, decocts 1.5 hours, for the second time add 6 times of water gagings, decocted collecting decoction 1.5 hours, filter, filtrate and the merging of above-mentioned aqueous solution are concentrated into relative density and are 1.25～1.30(50～55 ℃) clear cream.1 part of qinghuo reagent, 2.5 parts of sucrose, 2 parts in dextrin and appropriate amount of ethanol, granulation, drying adds the volatile oil of above-mentioned radix bupleuri, Radix Angelicae Sinensis, and mixing is made 1000g, and get final product.Every packed 6g; Boiling water is taken after mixing it with water, a 6g, 2 times on the one.
Rough gentian rushes down the heat of the moon of fainting in the side, is monarch drug in a prescription, and the heat of the root of large-flowered skullcap, cape jasmine clearing lung-heat and three warmers to be helping it, and rhizoma alismatis rushes down that kidney channel is wet, and it is to be ministerial drug to help it that akebi, plantain seed are rushed down the wet of small intestine bladder, so medicines of dropping of bitter cold all.So with Radix Angelicae Sinensis, the dried rhizome of rehmannia to nourish blood tonifying liver, be adjutant altogether, with Radix Glycyrrhizae with in slow and do not make and injure one's stomach, for making medicine also.
Only has the discriminating to the ministerial drug cape jasmine in the original detection method of Longdanxiegan particle, but the monarch drug in a prescription role also is very important in the well-known side, also have other ministerial drug such as the root of large-flowered skullcap, and unique medicine Radix Glycyrrhizae that makes also plays an important role, as do not have corresponding detection means to cause easily detection method with larger one-sidedness, so only content that can not comprehensively reflect the important composition of Longdanxiegan particle to the discriminating of cape jasmine.The aristolochic acid that wherein contains as the akebi of ministerial drug simultaneously needs to remove in the preparation process, so also be not incomplete to this assay at present.
Summary of the invention
The invention provides a kind of detection method of Longdanxiegan particle active principle
,To solve the incomplete problem of the detection that exists in the present detection method.
The technical scheme that the present invention takes is to comprise following discrimination method and content assaying method and inspection method:
(1) discrimination method
(1) get this product Longdanxiegan particle 12g, add water 30ml, heating makes dissolving, let cool, use extracted by ether 3 times, each 15ml, discard ether solution, the full normal butyl alcohol that closes of water extracts 3 times again, each 15ml, merge normal butyl alcohol liquid, add the equal-volume ammonia solution, shake up, place layering, divide and get supernatant, the recovered under reduced pressure normal butyl alcohol is to doing, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Gardenoside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take ethyl acetate: acetone: formic acid: water=5:5:1:1 launches as developping agent, takes out, dry, spray is with ethanol solution of sulfuric acid, and is 110 ℃ of bakings 10 minutes, clear to the spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
(2) get this product Longdanxiegan particle 12g, porphyrize adds methyl alcohol 50ml, add hot reflux 1 hour, and filtered the filtrate evaporate to dryness, residue adds water 15ml, and heating makes dissolving, lets cool, the full normal butyl alcohol that closes of water extracts 2 times, each 15ml merges n-butanol extracting liquid, and the recovered under reduced pressure normal butyl alcohol is to doing, residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take ethyl acetate: butanone: formic acid: water=5:3:1.1:1 is as developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
(3) get need testing solution under above-mentioned (2), as need testing solution; Extracting Radix Glycyrrhizae control medicinal material 2g adds methyl alcohol 20ml in addition, adds hot reflux 30 minutes, filters, and filtrate is concentrated into 2ml, in contrast medicinal material solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, take ethyl acetate: formic acid: glacial acetic acid: water=15:1:1:2 is as developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(4) get above-mentioned (2) lower need testing solution, be added on the neutral alumina column of having handled well, the neutral alumina that fills 120 orders, 2g, column internal diameter 15mm, usefulness methyl alcohol 20ml wash-out, eluent is concentrated into 2ml, as need testing solution; Other gets the gentiamarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 3 μ l of above-mentioned two kinds of solution, put in same silica G F respectively
254On the thin layer plate, with chloroform: methyl alcohol: water=13:6:2, lower floor's solution of placing below 10 ℃ is developping agent, launches, and takes out, and dries, and puts under the 254nm ultraviolet lamp inspection and knows; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
(2) content assaying method
(1) according to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: water=20:80 as mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the gentiamarin peak should be not less than 3000;
The preparation of reference substance solution: it is an amount of to get the gentiamarin reference substance, accurately weighed, adds methyl alcohol and makes the solution that every 1ml contains 25 μ g, and get final product;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, porphyrize is got 1g, accurately weighed, put in the tool plug conical flask the accurate methyl alcohol 25ml that adds, close plug, weighed weight, ultrasonic processing 30 minutes, power is 250W, and frequency is 50kHz, lets cool, weighed weight is supplied the weight that subtracts mistake, mixing with methyl alcohol again, filter, get subsequent filtrate, and get final product;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product;
(2) preparation of reference substance solution: precision takes by weighing at 4 hours scutelloside reference substance of 60 ℃ of drying under reduced pressure an amount of, adds methyl alcohol and makes the solution that every 1ml contains 60 μ l, and get final product;
The need testing solution preparation: get this product Longdanxiegan particle under the content uniformity item, fully porphyrize is got 1.5g, accurately weighed, put in the 50ml measuring bottle, add 60% methyl alcohol 45ml, ultrasonic processing, 30 minutes, power 150W, frequency 25KHz lets cool to room temperature, adds 60% methyl alcohol to scale, shake up, filter, discard just filtrate, get subsequent filtrate, and get final product;
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Methyl alcohol: water: phosphoric acid=47:53:0.2 is mobile phase; The detection wavelength is 280nm; Number of theoretical plate is pressed the scutelloside peak and is calculated, and should be not less than 2500;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatography liquid, measures, and get final product;
(3) preparation of reference substance solution: it is an amount of to get the Gardenoside reference substance, accurately weighed, adds 50% methyl alcohol and makes the solution that every 1ml contains 30 μ g, and get final product;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, fully porphyrize is got 1.5g, accurately weighed, put in the tool plug conical flask the accurate 50% methyl alcohol 50ml that adds, weighed weight, ultrasonic processing 20 minutes, power is 300W, frequency is 50kHz, let cool, weighed weight is supplied the weight that subtracts mistake with 50% methyl alcohol again, shake up, filter; Precision is measured subsequent filtrate 10ml, puts in the 25ml measuring bottle, adds 50% methyl alcohol to scale, shakes up, and filters, and gets subsequent filtrate, and get final product;
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile: water=11:89 as mobile phase; The detection wavelength is 238nm, and number of theoretical plate calculates by the Gardenoside peak should be not less than 2000;
Determination method is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and be get final product;
(4) according to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: 0.2mol/L ammonium acetate solution: glacial acetic acid=67:33:1 is as mobile phase; The detection wavelength is 250nm; Number of theoretical plate calculates by the glycyrrhizic acid peak should be not less than 2000;
The preparation of reference substance solution: extracting Radix Glycyrrhizae acid ammonium reference substance 10mg, accurately weighed, the mixed solution that adds 0.5% ammonia solution: methyl alcohol=1:1 is made the solution that every 1ml contains 0.2mg, is equivalent to every 1ml and contains glycyrrhizic acid 0.1959mg, and get final product;
The preparation of need testing solution
:Get this product Longdanxiegan particle under the content uniformity item, porphyrize is got 1.5g, accurately weighed, put in the tool plug conical flask the accurate mixed solution 50ml that adds 0.5% ammonia solution: methyl alcohol=1:1, close plug, weighed weight, ultrasonic processing 30 minutes, power is 250W, and frequency is 20kHz, lets cool, weighed weight is supplied the weight that subtracts mistake with above-mentioned mixed solution again, shakes up, filter, get subsequent filtrate, and get final product;
Determination method
Precision is drawn reference substance solution and each 20 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product;
(3) inspection method:
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability
:Be filling agent with octadecyl silane; Methyl alcohol: water: glacial acetic acid=57:43:1 is mobile phase; The detection wavelength is 317nm, and theoretical cam curve is calculated by the aristolochic acid A peak should be not less than 8000;
The preparation of reference substance solution: precision takes by weighing aristolochic acid A reference substance 5mg, puts in the 25ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and the accurate 0.5ml that draws puts in the 100ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and get final product, and it is 1 μ g that every 1ml contains aristolochic acid A;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, mixing, porphyrize, get 5g, accurately weighed, put in the tool plug conical flask, accurate methyl alcohol: formic acid=98:2,50ml, the weighed weight of adding, ultrasonic processing 30 minutes, power 250W, frequency 40kHz, take out, let cool, more weighed weight, supply with methyl alcohol and to subtract weight loss, shake up, filter, the accurate subsequent filtrate 10ml that draws, evaporate to dryness, residue add water 20ml makes dissolving, hydrochloric acid solution adjust pH 2-3 with 10% uses extracted by ether 4 times, each 10ml, merge ether extracted liquid, put in 60 ℃ of water-baths and volatilize, residue adds a small amount of methyl alcohol dissolving and is transferred in the 10ml measuring bottle, adds methyl alcohol and is diluted to scale, shake up, filter, get subsequent filtrate, and get final product;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product.
Advantage of the present invention is: standard the detection method of Longdanxiegan particle, on original basis that cape jasmine is differentiated, by setting up discriminating and the assay to the monarch drug in a prescription rough gentian, and to the ministerial drug root of large-flowered skullcap, the discriminating that makes the medicine Radix Glycyrrhizae and assay, and to ministerial drug cape jasmine and assay, more fully the composition of detection of drugs and its compatibility are understood its content and stability, also help to illustrate the medical substance basis of medicine; Increase simultaneously the inspection to aristolochic acid, more be conducive to reduce its toxic and side effect.
Embodiment
The Longdanxiegan particle is provided by prescription and explained hereafter in the background technology by JiLin ZiXin Pharmacy Co., Ltd.
Comprise following discrimination method and content assaying method and inspection method:
(1) discrimination method
(1) get this product Longdanxiegan particle 12g, add water 30ml, heating makes dissolving, let cool, use extracted by ether 3 times, each 15ml, discard ether solution, the full normal butyl alcohol that closes of water extracts 3 times again, each 15ml, merge normal butyl alcohol liquid, add the equal-volume ammonia solution, shake up, place layering, divide and get supernatant, the recovered under reduced pressure normal butyl alcohol is to doing, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Gardenoside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take ethyl acetate: acetone: formic acid: water=5:5:1:1 launches as developping agent, takes out, dry, spray is with ethanol solution of sulfuric acid, and is 110 ℃ of bakings 10 minutes, clear to the spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
(2) get this product Longdanxiegan particle 12g, porphyrize adds methyl alcohol 50ml, add hot reflux 1 hour, and filtered the filtrate evaporate to dryness, residue adds water 15ml, and heating makes dissolving, lets cool, the full normal butyl alcohol that closes of water extracts 2 times, each 15ml merges n-butanol extracting liquid, and the recovered under reduced pressure normal butyl alcohol is to doing, residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take ethyl acetate: butanone: formic acid: water=5:3:1.1:1 is as developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
(3) get need testing solution under above-mentioned (2), as need testing solution; Extracting Radix Glycyrrhizae control medicinal material 2g adds methyl alcohol 20ml in addition, adds hot reflux 30 minutes, filters, and filtrate is concentrated into 2ml, in contrast medicinal material solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, take ethyl acetate: formic acid: glacial acetic acid: water=15:1:1:2 is as developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(4) get above-mentioned (2) lower need testing solution, be added on the neutral alumina column of having handled well, the neutral alumina that fills 120 orders, 2g, column internal diameter 15mm, usefulness methyl alcohol 20ml wash-out, eluent is concentrated into 2ml, as need testing solution; Other gets the gentiamarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 3 μ l of above-mentioned two kinds of solution, put in same silica G F respectively
254On the thin layer plate, with chloroform: methyl alcohol: water=13:6:2, lower floor's solution of placing below 10 ℃ is developping agent, launches, and takes out, and dries, and puts under the 254nm ultraviolet lamp inspection and knows; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
(2) content assaying method
(1) according to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: water=20:80 as mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the gentiamarin peak should be not less than 3000;
The preparation of reference substance solution: it is an amount of to get the gentiamarin reference substance, accurately weighed, adds methyl alcohol and makes the solution that every 1ml contains 25 μ g, and get final product;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, porphyrize is got 1g, accurately weighed, put in the tool plug conical flask the accurate methyl alcohol 25ml that adds, close plug, weighed weight, ultrasonic processing 30 minutes, power is 250W, and frequency is 50kHz, lets cool, weighed weight is supplied the weight that subtracts mistake, mixing with methyl alcohol again, filter, get subsequent filtrate, and get final product;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product;
(2) preparation of reference substance solution: precision takes by weighing at 4 hours scutelloside reference substance of 60 ℃ of drying under reduced pressure an amount of, adds methyl alcohol and makes the solution that every 1ml contains 60 μ l, and get final product;
The need testing solution preparation: get this product Longdanxiegan particle under the content uniformity item, fully porphyrize is got 1.5g, accurately weighed, put in the 50ml measuring bottle, add 60% methyl alcohol 45ml, ultrasonic processing, 30 minutes, power 150W, frequency 25KHz lets cool to room temperature, adds 60% methyl alcohol to scale, shake up, filter, discard just filtrate, get subsequent filtrate, and get final product;
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Methyl alcohol: water: phosphoric acid=47:53:0.2 is mobile phase; The detection wavelength is 280nm; Number of theoretical plate is pressed the scutelloside peak and is calculated, and should be not less than 2500;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatography liquid, measures, and get final product;
(3) preparation of reference substance solution: it is an amount of to get the Gardenoside reference substance, accurately weighed, adds 50% methyl alcohol and makes the solution that every 1ml contains 30 μ g, and get final product;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, fully porphyrize is got 1.5g, accurately weighed, put in the tool plug conical flask the accurate 50% methyl alcohol 50ml that adds, weighed weight, ultrasonic processing 20 minutes, power is 300W, frequency is 50kHz, let cool, weighed weight is supplied the weight that subtracts mistake with 50% methyl alcohol again, shake up, filter; Precision is measured subsequent filtrate 10ml, puts in the 25ml measuring bottle, adds 50% methyl alcohol to scale, shakes up, and filters, and gets subsequent filtrate, and get final product;
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile: water=11:89 as mobile phase; The detection wavelength is 238nm, and number of theoretical plate calculates by the Gardenoside peak should be not less than 2000;
Determination method is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and be get final product;
(4) according to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: 0.2mol/L ammonium acetate solution: glacial acetic acid=67:33:1 is as mobile phase; The detection wavelength is 250nm; Number of theoretical plate calculates by the glycyrrhizic acid peak should be not less than 2000;
The preparation of reference substance solution: extracting Radix Glycyrrhizae acid ammonium reference substance 10mg, accurately weighed, the mixed solution that adds 0.5% ammonia solution: methyl alcohol=1:1 is made the solution that every 1ml contains 0.2mg, is equivalent to every 1ml and contains glycyrrhizic acid 0.1959mg, and get final product;
The preparation of need testing solution
:Get this product Longdanxiegan particle under the content uniformity item, porphyrize is got 1.5g, accurately weighed, put in the tool plug conical flask the accurate mixed solution 50ml that adds 0.5% ammonia solution: methyl alcohol=1:1, close plug, weighed weight, ultrasonic processing 30 minutes, power is 250W, and frequency is 20kHz, lets cool, weighed weight is supplied the weight that subtracts mistake with above-mentioned mixed solution again, shakes up, filter, get subsequent filtrate, and get final product;
Determination method
Precision is drawn reference substance solution and each 20 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product;
(3) inspection method:
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability
:Be filling agent with octadecyl silane; Methyl alcohol: water: glacial acetic acid=57:43:1 is mobile phase; The detection wavelength is 317nm, and theoretical cam curve is calculated by the aristolochic acid A peak should be not less than 8000;
The preparation of reference substance solution: precision takes by weighing aristolochic acid A reference substance 5mg, puts in the 25ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and the accurate 0.5ml that draws puts in the 100ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and get final product, and it is 1 μ g that every 1ml contains aristolochic acid A;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, mixing, porphyrize, get 5g, accurately weighed, put in the tool plug conical flask, accurate methyl alcohol: formic acid=98:2,50ml, the weighed weight of adding, ultrasonic processing 30 minutes, power 250W, frequency 40kHz, take out, let cool, more weighed weight, supply with methyl alcohol and to subtract weight loss, shake up, filter, the accurate subsequent filtrate 10ml that draws, evaporate to dryness, residue add water 20ml makes dissolving, hydrochloric acid solution adjust pH 2-3 with 10% uses extracted by ether 4 times, each 10ml, merge ether extracted liquid, put in 60 ℃ of water-baths and volatilize, residue adds a small amount of methyl alcohol dissolving and is transferred in the 10ml measuring bottle, adds methyl alcohol and is diluted to scale, shake up, filter, get subsequent filtrate, and get final product;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product.
Claims (1)
1. the detection method of a Longdanxiegan particle active principle comprises the discrimination method of cape jasmine:
Get this product Longdanxiegan particle 12g, add water 30ml, heating makes dissolving, let cool, use extracted by ether 3 times, each 15ml, discard ether solution, the full normal butyl alcohol that closes of water extracts 3 times again, each 15ml, merge normal butyl alcohol liquid, add the equal-volume ammonia solution, shake up, place layering, divide and get supernatant, the recovered under reduced pressure normal butyl alcohol is to doing, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the Gardenoside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take ethyl acetate: acetone: formic acid: water=5:5:1:1 launches as developping agent, takes out, dry, spray is with ethanol solution of sulfuric acid, and is 110 ℃ of bakings 10 minutes, clear to the spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
Characterized by further comprising following discrimination method and content assaying method and inspection method:
(1) discrimination method
(1) get this product Longdanxiegan particle 12g, porphyrize adds methyl alcohol 50ml, add hot reflux 1 hour, and filtered the filtrate evaporate to dryness, residue adds water 15ml, and heating makes dissolving, lets cool, the full normal butyl alcohol that closes of water extracts 2 times, each 15ml merges n-butanol extracting liquid, and the recovered under reduced pressure normal butyl alcohol is to doing, residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and makes the solution that every 1ml contains 5mg, in contrast product solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, take ethyl acetate: butanone: formic acid: water=5:3:1.1:1 is as developping agent, launch, take out, dry, spray is with 5% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
(2) get need testing solution under above-mentioned (1), as need testing solution; Extracting Radix Glycyrrhizae control medicinal material 2g adds methyl alcohol 20ml in addition, adds hot reflux 30 minutes, filters, and filtrate is concentrated into 2ml, in contrast medicinal material solution; Test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively on the silica gel g thin-layer plate of same usefulness 1% sodium hydroxide solution preparation, take ethyl acetate: formic acid: glacial acetic acid: water=15:1:1:2 is as developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) get above-mentioned (1) lower need testing solution, be added on the neutral alumina column of having handled well, the neutral alumina that fills 120 orders, 2g, column internal diameter 15mm, usefulness methyl alcohol 20ml wash-out, eluent is concentrated into 2ml, as need testing solution; Other gets the gentiamarin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the test of an appendix VI of Chinese Pharmacopoeia version in 2005 B thin-layered chromatography, draw each 3 μ l of above-mentioned two kinds of solution, put in same silica G F respectively
254On the thin layer plate, with chloroform: methyl alcohol: water=13:6:2, lower floor's solution of placing below 10 ℃ is developping agent, launches, and takes out, and dries, and puts under the 254nm ultraviolet lamp inspection and knows; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;
(2) content assaying method
(1) according to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: water=20:80 as mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the gentiamarin peak should be not less than 3000;
The preparation of reference substance solution: it is an amount of to get the gentiamarin reference substance, accurately weighed, adds methyl alcohol and makes the solution that every 1ml contains 25 μ g, and get final product;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, porphyrize is got 1g, accurately weighed, put in the tool plug conical flask the accurate methyl alcohol 25ml that adds, close plug, weighed weight, ultrasonic processing 30 minutes, power is 250W, and frequency is 50kHz, lets cool, weighed weight is supplied the weight that subtracts mistake, mixing with methyl alcohol again, filter, get subsequent filtrate, and get final product;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product;
(2) preparation of reference substance solution: precision takes by weighing at 4 hours scutelloside reference substance of 60 ℃ of drying under reduced pressure an amount of, adds methyl alcohol and makes the solution that every 1ml contains 60 μ l, and get final product;
The need testing solution preparation: get this product Longdanxiegan particle under the content uniformity item, fully porphyrize is got 1.5g, accurately weighed, put in the 50ml measuring bottle, add 60% methyl alcohol 45ml, ultrasonic processing, 30 minutes, power 150W, frequency 25KHz lets cool to room temperature, adds 60% methyl alcohol to scale, shake up, filter, discard just filtrate, get subsequent filtrate, and get final product;
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: be filling agent with octadecylsilane chemically bonded silica; Methyl alcohol: water: phosphoric acid=47:53:0.2 is mobile phase; The detection wavelength is 280nm; Number of theoretical plate is pressed the scutelloside peak and is calculated, and should be not less than 2500;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, injects liquid chromatography liquid, measures, and get final product;
(3) preparation of reference substance solution: it is an amount of to get the Gardenoside reference substance, accurately weighed, adds 50% methyl alcohol and makes the solution that every 1ml contains 30 μ g, and get final product;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, fully porphyrize is got 1.5g, accurately weighed, put in the tool plug conical flask the accurate 50% methyl alcohol 50ml that adds, weighed weight, ultrasonic processing 20 minutes, power is 300W, frequency is 50kHz, let cool, weighed weight is supplied the weight that subtracts mistake with 50% methyl alcohol again, shake up, filter; Precision is measured subsequent filtrate 10ml, puts in the 25ml measuring bottle, adds 50% methyl alcohol to scale, shakes up, and filters, and gets subsequent filtrate, and get final product;
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile: water=11:89 as mobile phase; The detection wavelength is 238nm, and number of theoretical plate calculates by the Gardenoside peak should be not less than 2000;
Determination method is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and be get final product;
(4) according to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol: 0.2mol/L ammonium acetate solution: glacial acetic acid=67:33:1 is as mobile phase; The detection wavelength is 250nm; Number of theoretical plate calculates by the glycyrrhizic acid peak should be not less than 2000;
The preparation of reference substance solution: extracting Radix Glycyrrhizae acid ammonium reference substance 10mg, accurately weighed, the mixed solution that adds 0.5% ammonia solution: methyl alcohol=1:1 is made the solution that every 1ml contains 0.2mg, is equivalent to every 1ml and contains glycyrrhizic acid 0.1959mg, and get final product;
The preparation of need testing solution
:Get this product Longdanxiegan particle under the content uniformity item, porphyrize is got 1.5g, accurately weighed, put in the tool plug conical flask the accurate mixed solution 50ml that adds 0.5% ammonia solution: methyl alcohol=1:1, close plug, weighed weight, ultrasonic processing 30 minutes, power is 250W, and frequency is 20kHz, lets cool, weighed weight is supplied the weight that subtracts mistake with above-mentioned mixed solution again, shakes up, filter, get subsequent filtrate, and get final product;
Determination method
Precision is drawn reference substance solution and each 20 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product;
(3) inspection method:
According to an appendix VI of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability
:Be filling agent with octadecyl silane; Methyl alcohol: water: glacial acetic acid=57:43:1 is mobile phase; The detection wavelength is 317nm, and theoretical cam curve is calculated by the aristolochic acid A peak should be not less than 8000;
The preparation of reference substance solution: precision takes by weighing aristolochic acid A reference substance 5mg, puts in the 25ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and the accurate 0.5ml that draws puts in the 100ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and get final product, and it is 1 μ g that every 1ml contains aristolochic acid A;
The preparation of need testing solution: get this product Longdanxiegan particle under the content uniformity item, mixing, porphyrize, get 5g, accurately weighed, put in the tool plug conical flask, accurate methyl alcohol: formic acid=98:2,50ml, the weighed weight of adding, ultrasonic processing 30 minutes, power 250W, frequency 40kHz, take out, let cool, more weighed weight, supply with methyl alcohol and to subtract weight loss, shake up, filter, the accurate subsequent filtrate 10ml that draws, evaporate to dryness, residue add water 20ml makes dissolving, hydrochloric acid solution adjust pH 2-3 with 10% uses extracted by ether 4 times, each 10ml, merge ether extracted liquid, put in 60 ℃ of water-baths and volatilize, residue adds a small amount of methyl alcohol dissolving and is transferred in the 10ml measuring bottle, adds methyl alcohol and is diluted to scale, shake up, filter, get subsequent filtrate, and get final product;
Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and the injection liquid chromatography is measured, and be get final product.
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