CN103575841B - Seven relieve internal heat clearly granule content detection method - Google Patents
Seven relieve internal heat clearly granule content detection method Download PDFInfo
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Abstract
The invention provides a kind of method using HPLC chromatography for simultaneous detection baicalin and emodin content, with the content detection of the particle that realizes relieving internal heat clearly to seven, the method comprises the following steps: the standard model and seven preparing baicalin and archen respectively relieves internal heat clearly the test sample of particle; By above-mentioned sample respectively through the process of HPLC chromatography, compare and calculate acquisition seven and to relieve internal heat clearly the content of baicalin in the test sample of particle and archen.Method of the present invention is to seven improvement of detection method of content relieving internal heat clearly particle in 2010 editions veterinary drug allusion quotations two, the content control method of method of the present invention is simple to operate, achieve effective constituent quantified controlling requirement in the tcm product to effective constituent kind complexity, the quality level of product is promoted, the standard method of content detection of the particle that can relieve internal heat clearly as new seven.
Description
Technical field
The present invention relates to a kind of seven to relieve internal heat clearly granule content detection method, refer to especially and a kind of method for measuring is carried out to seven content of baicalin and archen in particle that relieve internal heat clearly simultaneously.
Background technology
Seven particles that relieve internal heat clearly are a kind of common veterinary drug sides, have the effects such as clearing heat and detoxicating, dampness removing and diarrhea-relieving.Prescription is containing the root of large-flowered skullcap, giant knotweed, the Chinese bulbul, kuh-seng, Radix Isatidis, thick wood-fern rhizome, folium isatidis etc., be mainly used in diarrhea due to damp-heat, the white scour of chicken, simultaneously also for preventing and treating porcine reproductive and respiratory syndrome, Mild type HC, PCV-II, blue otopathy poison, influenza virus and fowl mild influenza, non typical newcastle disease, chronic respiratory tract disease, infective bronchitis, infectious laryngotracheitis, bursal disease, egg-decreasing syndrome, fowl enterotoxin syndrome etc., also have and strengthen the effect such as immunity, reduction stress reaction, and can be used as health-care good product use.Therefore, seven particles that relieve internal heat clearly commercially gain universal acceptance.Due to seven effects relieving internal heat clearly baicalin in granule medicament, archen, baicalin, emodin content are extremely important to seven relieve internal heat the clearly content detection of particle." People's Republic of China's veterinary drug allusion quotation " (2010 two) disclose and a kind ofly use thin-layered chromatography to carry out seven to relieve internal heat clearly the method that granule content detects, but this seven relieves internal heat clearly the quality standard of particle only to the discriminating of scutelloside, giant knotweed, archen, indigo and indigo red, there is no concrete assay item, therefore, seven granular masss that relieve internal heat clearly are controlled to be difficult to reach the standard of quantification.
Because seven particles that relieve internal heat clearly are a kind of veterinary drug herbal mixtures containing multi-component complex composition, due to complicated component, cause content wayward, the security of Chinese medicine and validity are affected.
Summary of the invention
In view of this, fundamental purpose of the present invention is to provide a kind of method using HPLC chromatography for simultaneous detection baicalin and emodin content, with the content detection of the particle that realizes relieving internal heat clearly to seven.
Namely the invention provides a kind of seven to relieve internal heat clearly granule content detection method, is use HPLC chromatography for simultaneous detection baicalin and emodin content.
Preferably, detection method of content of the present invention, comprises the following steps:
The standard model of A, respectively preparation baicalin and archen;
The test sample that B, preparation seven relieve internal heat clearly particle;
The test sample of C, the particle that to be relieved internal heat clearly by the standard model and seven of described baicalin and archen, respectively through the process of HPLC chromatography, compares and calculates acquisition seven and to relieve internal heat clearly the content of baicalin in the test sample of particle and archen.
Namely the present invention is respectively through the process of HPLC chromatography by the test sample of the particle that the standard model of baicalin and the standard model of archen and seven relieved internal heat clearly, after the typical curve of the standard model of the standard model and archen that obtain baicalin, the test sample that relieve internal heat particle clearly with seven contrasts through the chromatographic curve of HPLC chromatography process, to obtain seven content relieving internal heat clearly baicalin in the test sample of particle and archen.
In order to fully discharge seven test samples relieving internal heat clearly particle, can with an organic solvent extract; More preferably, ethanol is used to extract; More preferably, the ethanol of 70% is used to extract the test sample that seven relieve internal heat clearly particle; Also other any Solvent Extract methods that this area is conventional can be used.
In order to fully discharge seven test samples relieving internal heat clearly particle, the method for mechanical treatment can also be used to process the test sample that seven relieve internal heat clearly particle; Preferably, milled processed can be carried out to seven particles that relieve internal heat clearly; More preferably, ultrasonic vibration process can be carried out to seven particles that relieve internal heat clearly; The method of other mechanical treatments that this area also can be used to commonly use processes the test sample that seven relieve internal heat clearly particle.
Of the present invention seven relieve internal heat clearly in granule content detection method, employ HPLC chromatography to process relieve internal heat the clearly test sample of particle of the standard model of baicalin and the standard model of archen and seven, wherein, the chromatographic column used in HPLC chromatography is reverse-phase chromatographic column; Preferably, the chromatographic column used in HPLC chromatography is C18 post.
Preferably, the mobile phase used in described HPLC chromatography is acetonitrile-water-phosphoric acid.Preferably, in described HPLC chromatography, mobile phase ratio is acetonitrile-water-phosphoric acid=22:78:0.2.
Preferably, the flow velocity in described HPLC chromatography is 1ml/min; Determined wavelength: 270nm; Column temperature: 35 DEG C.
As seen from the above, the invention provides a kind of HPLC chromatography determination method, Simultaneously test seven relieves internal heat clearly baicalin in particle and emodin content, solves the seven content detection problems relieving internal heat clearly particle.
invention effect
The present invention, with reference to the detection method of the content of archen in 2010 editions veterinary drug allusion quotations, two baicalin in Scutellaria baicalensis Georgis and giant knotweed, suitably improves in conjunction with actual conditions, and through Method validation.This seven relieve internal heat clearly content control method of particle is simple to operate, achieves effective constituent quantified controlling requirement in the tcm product to effective constituent kind complexity, the quality level of product is promoted, has the prospect of commercial popularization.
Accompanying drawing explanation
Fig. 1 is the chromatogram of the standard model of baicalin and the standard model of archen;
Fig. 2 is the chromatogram of negative control;
Fig. 3 is seven chromatograms of test sample relieving internal heat clearly particle.
Embodiment
The C18 post used in the embodiment of the present invention refers to anti-phase ODS post (octadecylsilane chemically bonded silica filler, Octadecylsilyl, is called for short ODS post), pass through embodiments of the invention, those skilled in the art know all C18 posts and can be used for realizing the present invention, as waters
c18 chromatographic column etc. can realize the present invention.
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1HPLC method Simultaneously test seven relieves internal heat clearly the content of scutelloside and archen in particle
In the embodiment of the present invention, HPLC chromatographic condition is:
Chromatographic column: Agilent-C18(150mmX4.6mm, 5um).
Mobile phase: acetonitrile-water-phosphoric acid (22:78:0.2)
Flow velocity: 1ml/min; Determined wavelength: 270nm; Column temperature: 35 DEG C.
Prepared by reference substance solution: scutelloside reference substance and the archen reference substance of getting 60 DEG C of drying under reduced pressure 4h are in right amount each, accurately weighed, put in same 25ml measuring bottle, the ethanol adding 70% makes dissolving and is diluted to scale, shake up, make every milliliter of reference substance solution containing scutelloside 0.3mg, archen 20ug.
Prepared by need testing solution: the test sample getting porphyrize is about 0.2g, accurately weighed, puts in 25ml measuring bottle, adds the ethanol 20ml of 70%, after sonic oscillation extracts 15min, let cool to room temperature, the ethanol adding 70% is diluted to scale, shake up, filter, get subsequent filtrate as need testing solution.
Determination method: accurate absorption reference substance solution and each 10ul of need testing solution, injection liquid chromatography, record chromatogram, with external standard method by calculated by peak area, to obtain final product.
The chromatographic condition that content assaying method of the present invention adopts, sample treatment are new method.
1, instrument and reagent, sample
Instrument: Waterse2695 high performance liquid chromatograph; UV2489 UV-detector.
Reagent: methyl alcohol, second eyeball are chromatographically pure; Glacial acetic acid is pure for analyzing; Water is secondary redistilled water.
Reference substance: scutelloside (110715-201014), archen (110756-201110) are purchased from China Veterinery Drug Inspection Office
Sample: seven relieve internal heat clearly particle, are produced by Luoyang Huizhong Veterinary Medicine Co. Ltd..{ [prescription] root of large-flowered skullcap 100g giant knotweed 100g Chinese bulbul 80g kuh-seng 80g Radix Isatidis 100g thick wood-fern rhizome 60g folium isatidis 40g; 7 tastes more than [method for making], boiling 2 times, 2 hours first times, second time 1 hour, decocting liquid filters, and filtrate merges, less than 80 DEG C are evaporated to relative density is 1.30 ~ 1.35(55 DEG C), obtain clear cream, add appropriate sucrose and dextrin, mixing, make particle, drying, makes 560g, to obtain final product.}
2 chromatographic conditions:
Chromatographic column: Agilent-C18(150mmX4.6mm, 5um).
Mobile phase: acetonitrile-water-phosphoric acid (22:78:0.2)
Flow velocity: 1ml/min; Determined wavelength: 270nm; Column temperature: 35 DEG C.
The preparation of 3 solution:
1. the preparation of reference substance solution:
A. the scutelloside reference substance getting 60 DEG C of drying under reduced pressure 4h is appropriate, and accurately weighed, put in 25ml measuring bottle, the ethanol adding 70% makes dissolving and is diluted to scale, shakes up, and makes every milliliter of reference substance solution containing scutelloside 0.3mg.
B. the archen reference substance getting 60 DEG C of drying under reduced pressure 4h is appropriate, accurately weighed, adds methyl alcohol and makes every 1m1 about containing the reference substance solution of archen 20ug.
2. the preparation of need testing solution: the test sample getting porphyrize is about 0.2g, accurately weighed, puts in 25ml measuring bottle, adds the ethanol 20ml of 70%, after sonic oscillation extracts 15min, let cool to room temperature, the ethanol adding 70% is diluted to scale, shake up, filter, get subsequent filtrate as need testing solution.
3. the preparation of negative sample: get the sample not adding the root of large-flowered skullcap and giant knotweed, prepares by the preparation method of need testing solution, measures, the results are shown in Figure 2 by the chromatographic condition will put in 2.Comparing visible with reference substance collection of illustrative plates (Fig. 1), the position that scutelloside, archen peak occur in reference substance collection of illustrative plates is occurring there are no peak.
The preparation of 4 typical curves:
Get respectively and will put reference substance solution 5,10,20,30,50ul in 3, inject high performance liquid chromatograph, according to the chromatographic condition will putting 2, obtain the chromatogram of Fig. 1, measure peak area.To contrast sample size for horizontal ordinate, with integrating peak areas value for ordinate, drawing standard curve, obtains regression equation scutelloside: y=93186x+25150, R
2=0.9993; Archen: y=6021.2x-22.043, R
2=0.9991.Result shows that scutelloside is at 1.5 ~ 15ug, and archen is good in 100 ~ 1000ng scope internal linear relation.
5 precision tests:
Get seven to relieve internal heat clearly particle 1 part, by under will putting 3 2. in method prepare test liquid, get test liquid 10ul, measure by above-mentioned chromatographic condition, result is as Fig. 3.Continuous repetition sample introduction 6 times, calculate the content of scutelloside and archen with external standard method, result records RSD and is respectively 1.34% and 2.16%.
6 determination of recovery rates:
Get oneself and know that seven of content relieve internal heat clearly particle 6 parts, accurately weighed, respectively to wherein add scutelloside reference substance solution 10ml, archen reference substance solution 10ml by under 3 2. in method prepare test liquid, under will putting the chromatographic condition of 2, measure respective content, calculate the recovery.Result average recovery rate is respectively 98.05%, 97.94%, RSD and is respectively 1.93%, 2.08%.
7 sample determinations:
Get 12 lot numbers seven prepared by same process respectively to relieve internal heat clearly particulate samples (lot number: 100501,100502,100503,100504,100601,100602,100603,100604,100702,100703,100704,100801 Luoyang Huizhong Veterinary Medicine Co. Ltd. produce), by under 3 2. in method prepare test liquid, accurate absorption reference substance solution and need testing solution 10ul respectively, by above-mentioned chromatographic condition, measure, the content of scutelloside and archen in calculation sample.Through carrying out scutelloside and emodin content mensuration to 12 batch samples, result is as following table 1.Wherein scutelloside mxm. is 17.186mg/g, and minimum is 14.534mg/g, and archen mxm. is 1.307mg/g, and minimum is 1.009mg/g.Limitation according to scutelloside in Milkvetch Root is 9.0%, and in Rhizoma Polygoni Cuspidati, the limitation of archen is 0.6%, and the theoretical content calculating scutelloside in this product is 16.07mg/g, and archen is 1.07mg/g.Consider the loss in large-scale production process, the limit of the content of scutelloside in this product be decided to be in every gram of particle and be no less than 12.5mg, archen is 0.8mg/g, to control inherent quality.
Table 1 uses method of the present invention to the measurement result of 12 batch samples
| Sample lot number | Content of baicalin (mg/g) | Emodin content (mg/g) |
| 100501 | 15.934 | 1.211 |
| 100502 | 16.066 | 1.094 |
| 100503 | 17.186 | 1.224 |
| 100504 | 16.873 | 1.307 |
| 100601 | 16.107 | 1.193 |
| 100602 | 16.996 | 1.067 |
| 100603 | 17.008 | 1.115 |
| 100604 | 15.817 | 1.034 |
| 100702 | 14.971. | 1.009 |
| 100703 | 14.534 | 1.108 |
| 100704 | 15.097 | 1.069 |
| 100801 | 15.737 | 1.294 |
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. seven to relieve internal heat clearly a granule content detection method, it is characterized in that, described method is for using HPLC chromatography for simultaneous detection baicalin and emodin content, wherein, in described HPLC chromatography, mobile phase ratio is acetonitrile-water-phosphoric acid=22:78:0.2, determined wavelength: 270nm, column temperature: 35 DEG C.
2. method according to claim 1, is characterized in that, comprises the following steps:
The standard model of A, respectively preparation baicalin and archen;
The test sample that B, preparation seven relieve internal heat clearly particle;
The test sample of C, the particle that to be relieved internal heat clearly by the standard model and seven of described baicalin and archen, respectively through the process of HPLC chromatography, compares and calculates acquisition seven and to relieve internal heat clearly the content of baicalin in the test sample of particle and archen.
3. method according to claim 2, is characterized in that, relieve internal heat the clearly test sample of particle of seven described in described step B is use the process of ultrasonic vibration method.
4. method according to claim 2, is characterized in that, uses chromatographic column for reversed-phase column in the HPLC chromatography described in described step C.
5. method according to claim 2, is characterized in that, uses chromatographic column for C18 post in the HPLC chromatography described in described step C.
6. method according to claim 2, is characterized in that, the flow velocity in the HPLC chromatography described in described step C is 1ml/min.
7. the method according to claim 1-6 any one, is characterized in that, relieve internal heat the clearly test sample of particle of the standard model of the baicalin described in described step C and the standard model of archen and seven is use identical HPLC chromatographic condition process.
8. the purposes that the method according to claim 1-7 any one relieves internal heat clearly in granular mass control seven.
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| CN1698757A (en) * | 2005-05-09 | 2005-11-23 | 贵阳云岩西创药物科技开发有限公司 | Pharmaceutical preparation for treating upper respiratory tract infection and its preparation process and quality control method |
| CN1876039A (en) * | 2005-05-09 | 2006-12-13 | 贵阳云岩西创药物科技开发有限公司 | Pharmaceutical composition for treating upper respiratory tract infection, its preparation process and quality control method |
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| CN1698757A (en) * | 2005-05-09 | 2005-11-23 | 贵阳云岩西创药物科技开发有限公司 | Pharmaceutical preparation for treating upper respiratory tract infection and its preparation process and quality control method |
| CN1876039A (en) * | 2005-05-09 | 2006-12-13 | 贵阳云岩西创药物科技开发有限公司 | Pharmaceutical composition for treating upper respiratory tract infection, its preparation process and quality control method |
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