CN104122340A - Method for determining contents of purple bergenia herb and liquorice in traditional Chinese medicine composition Anerning preparation - Google Patents
Method for determining contents of purple bergenia herb and liquorice in traditional Chinese medicine composition Anerning preparation Download PDFInfo
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Abstract
The invention discloses a method for determining contents of purple bergenia herb and liquorice in traditional Chinese composition Anerning preparation. The method is characterized in that a high-performance liquid chromatography is adopted, and one chromatographic condition is used for simultaneously determining the content of purple bergenia herb and the content of liquorice in the traditional Chinese medicine composition Anerning preparation containing the purple bergenia herb and liquorice, so that convenience in quality control and simplicity and convenience in operation can be achieved, and the analysis data is accurate. By adopting the method, not only can the analysis time of the analysis personnel be saved and the working efficiency be increased, but also the safety, effectiveness and controllability of the preparation can be improved.
Description
Technical field
The present invention relates to a kind of content assaying method of compound Chinese medicinal preparation, particularly purple bergenia herb, Radix Glycyrrhizae content assaying method in the peaceful preparation of a kind of peace youngster, belong to medical technical field.
Background technology
The exclusive product of peace youngster peaceful particle Wei Jinhe Tibetan medicine incorporated company, standard number: WS-10640(ZD-0640)-2002-2012Z.The peaceful particle of peace youngster is to be made by the medicinal material of following weight proportion: Tabasheer 66.7g, safflower 53.3g, calculus bovis factitius 5.3g, purple bergenia herb 53.3g, Radix Glycyrrhizae 53.3g, high mountain horseradish dish 53.3g, Lagotis brachystachya Maxim 66.7g, santal 66.7g, Tangut aconite 66.7g.There is removing heat to expel wind, the effects such as preventing phlegm from forming and stopping coughing.For children's wind-heat cold, bring up phlegm when one coughs, heating pharyngalgia, the infection of the upper respiratory tract is shown in above-mentioned disease person.Due to its good curative effect, be now widely used in clinical.
Medicine initial quality is controlled the quality of only controlling calculus bovis factitius, and method is simple coarse extremely, at all can not the production run of comprehensive and accurate this compound preparation of reflection and the quality of control medicine.Radix Glycyrrhizae and purple bergenia herb all have good antibechic, resolve phlegm effect, are main ingredient in side.Radix Glycyrrhizae and purple bergenia herb are used as medicine after being extraction, by assay, to detect quality in its side, can better control extraction process, and then guarantee the safe, effective, quality controllable of product quality.
Control respectively the quality of Radix Glycyrrhizae and purple bergenia herb, need use two kinds of methods to measure, through retrieval, do not find to measure at present document and the patented invention of purple bergenia herb and Radix Glycyrrhizae content simultaneously.
Summary of the invention
For the deficiencies in the prior art, the invention provides purple bergenia herb, Radix Glycyrrhizae content assaying method in the peaceful preparation of a kind of peace youngster.
Summary of the invention
The present invention uses a chromatographic condition purple bergenia herb, Radix Glycyrrhizae in the peaceful preparation of peace youngster to be carried out to quality control simultaneously, easy and simple to handle, analyzes data accurate.Both save analyst's analysis time, improved work efficiency, improved again security, validity and the controllability of preparation simultaneously.
Technical scheme of the present invention is as follows:
Purple bergenia herb, a Radix Glycyrrhizae content assaying method in the peaceful preparation of peace youngster, step is as follows:
According to high performance liquid chromatography (appendix VI D of < < Chinese Pharmacopoeia > > version in 2010), measure;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, and the volume parts of take is Mobile phase B than 0.05% phosphoric acid solution, adopts gradient elution mode: 0min → 20min → 30min → 60min, acetonitrile: 7% → 7% → 36% → 36%; Volume parts is than 0.05% phosphoric acid solution: 93% → 93% → 64% → 64%, and detection wavelength is 245nm-275nm; Number of theoretical plate calculates and should be not less than 3000 by glycyrrhizic acid peak;
The preparation of reference substance solution: get Bergenin reference substance, ammonium glycyrrhetate reference substance, add methyl alcohol and make every 1ml containing the solution of Bergenin, each 50 μ g of ammonium glycyrrhetate, obtain;
The preparation of need testing solution: solid is pacified after the peaceful preparation porphyrize of youngster, get powder 4g, accurately weighed, put in the conical flask of tool plug, add methyl alcohol 25ml, close plug, ultrasonic processing 30-40min, lets cool, and supplies the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, obtain;
Determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains.
The peaceful preparation of described peace youngster refers to and contains purple bergenia herb and Radix Glycyrrhizae is main ingredient; contain the multiple or whole of Tabasheer, safflower, calculus bovis factitius, high mountain horseradish dish, Lagotis brachystachya Maxim, santal, Tangut aconite simultaneously; technique routinely; add conventional auxiliary material to be prepared into clinical acceptable any formulation, comprise capsule, pill, micropill, dripping pill, tablet, capsule, particle, medicine materical crude slice or dispersing tablet.
Described gradient elution mode is gradient changes component (composition, ionic strength etc.) or the pH of eluent, to the method that the rational time wash-in of components different on chromatographic column is taken off.
Preferred according to the present invention; bulk drug consists of the Chinese medicine composition of Tabasheer 66.7g, safflower 53.3g, calculus bovis factitius 5.3g, purple bergenia herb 53.3g, Radix Glycyrrhizae 53.3g, high mountain horseradish dish 53.3g, Lagotis brachystachya Maxim 66.7g, santal 66.7g, Tangut aconite 66.7g and pacifies purple bergenia herb, a Radix Glycyrrhizae content assaying method in the peaceful particle of youngster, and step is as follows:
According to high performance liquid chromatography (appendix VI D of < < Chinese Pharmacopoeia > > version in 2010), measure;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, and the volume parts of take is Mobile phase B than 0.05% phosphoric acid solution, adopts gradient elution mode: 0min → 20min → 30min → 60min, acetonitrile: 7% → 7% → 36% → 36%; Volume parts is than 0.05% phosphoric acid solution: 93% → 93% → 64% → 64%, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 3000 by glycyrrhizic acid peak;
The preparation of reference substance solution: get Bergenin reference substance, ammonium glycyrrhetate reference substance, add methyl alcohol and make every 1ml containing the solution of Bergenin, each 50 μ g of ammonium glycyrrhetate, obtain;
The preparation of need testing solution: will pacify after the peaceful particle porphyrize of youngster, get powder 4g, accurately weighed, put in the conical flask of tool plug, add methyl alcohol 25ml, close plug, ultrasonic processing 30min, lets cool, and supplies the weight of less loss with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, obtains;
Determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In the peaceful particle of peace youngster, the content of purple bergenia herb, in Bergenin glycosides, must not be less than 0.7mg/g; Radix Glycyrrhizae content, in glycyrrhizic acid, must not be less than 0.3mg/g.
The present invention uses a chromatographic condition the peaceful preparation of the peace youngster of containing purple bergenia herb and Radix Glycyrrhizae to be carried out to quality control simultaneously, easy and simple to handle, analyzes data accurate.Both save analyst's analysis time, improved work efficiency, improved security, validity and the controllability of preparation simultaneously.For example pacify the content assaying method that only has calculus bovis factitius under the peaceful granular raw quality standard of youngster item, can not effectively detect the content of other principal ingredient purple bergenia herbs, Radix Glycyrrhizae.The present invention has carried out corresponding raising to the peaceful granular mass standard of existing peace youngster, use a chromatographic condition purple bergenia herb and Radix Glycyrrhizae to be carried out to assay simultaneously, improved work efficiency, improve security, validity and the controllability of preparation simultaneously, thereby guaranteed the healthy of its clinical efficacy and extensive patients.
Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.
Experimental example 1 assay experiment
1. instrument, reagent and test sample
Instrument: the L-2100 of Hitachi pump, the L-2400 of Hitachi UV-detector, Shimadzu AUW-220D type electronic balance.
Reference substance: Bergenin reference substance (Chinese pharmaceutical biological product is identified institute) lot number: 111532-200202, ammonium glycyrrhetate reference substance (Products in China is identified institute) lot number: 110731-200615.
Sample: the peace peaceful particle of youngster (Jin He Tibetan medicine incorporated company provides), lot number: 20121110,20121114,20121119.
2. detect the selection of wavelength
Bergenin has maximum absorption band at 275nm place; Ammonium glycyrrhetate has maximum absorption band at 250nm place.In preparation, Determination of Bergenin doubles compared with glycyrrhizic acid content, therefore select Bergenin maximum absorption band 250nm for detecting wavelength.
3. mobile phase is selected
Bergenin and glycyrrhizic acid polarity differ larger, adopt isocratic elution to be difficult to two kinds of compositions to measure simultaneously, therefore select gradient elution to carry out chromatographic resolution.
Discovery while using methanol-water system to carry out chromatographic resolution, easily forms larger gradient peak at 250nm carapax et plastruw testudinis alcohol-water system, and peak is poor simultaneously, and impact is measured.
Consider the tolerance of chromatographic column, and the stability of sample is while being used acetonitrile-0.05% Phosphoric Acid to carry out gradient elution, above problem is resolved.
4, the selection of gradient elution time
While finding to carry out wash-out by the following time by great many of experiments, purple bergenia herb and glycyrrhizic acid all can reach good testing requirement.Mobile phase A is acetonitrile, and Mobile phase B is 0.05% phosphoric acid.In Table 1 and accompanying drawing 1, accompanying drawing 2.
Table 1 gradient elution table
5. the preparation of typical curve and the investigation of linear relationship
Precision measures Bergenin reference substance stock solution solution (112.5 μ g/ml) and ammonium glycyrrhetate reference substance stock solution solution (108.6 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put respectively in 10ml volumetric flask, methyl alcohol is released to scale, shake up, each accurate sample introduction 10 μ l, the peak area (A) of take is ordinate, reference substance concentration (C) is horizontal ordinate, carry out linear regression, in Table 2, table 3 and Fig. 3, Fig. 4;
Table 2 purple bergenia herb linear relationship is investigated result
| Sequence number | Concentration C (μ g/ml) | Peak area A (μ Vs) |
| 1 | 11.25 | 10912 |
| 2 | 33.75 | 34628 |
| 3 | 56.25 | 56139 |
| 4 | 90.00 | 87854 |
| 5 | 112.5 | 110569 |
Regression equation: A=974.87C+796.79
Related coefficient: R=0.9998
Table 3 glycyrrhizic acid linear relationship is investigated result
| Sequence number | Concentration C (μ g/ml) | Peak area A (μ Vs) |
| 1 | 10.64 | 10801 |
| 2 | 31.92 | 33713 |
| 3 | 53.20 | 53628 |
| 4 | 85.12 | 90427 |
| 5 | 106.40 | 110038 |
Regression equation: A=1044.8C-307.74
Related coefficient: R=0.9995
Result shows: Bergenin is within the scope of 11.25 μ g/ml-112.50 μ g/ml, and linear relationship is good; Glycyrrhizic acid is within the scope of 10.64-106.40 μ g/ml, and linear relationship is good.
6. precision test
Accurate Bergenin and the ammonium glycyrrhetate reference substance solution 10 μ l of drawing, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, records peak area and calculates relative standard deviation.In Table 4, table 5.
Table 4 Bergenin Precision test result
Table 5 glycyrrhizic acid Precision test result
Note: A represents peak area;
represent average peak area; RSD represents relative standard deviation.
Result shows: instrument precision is good.
7. stability test
After prepared by need testing solution, the accurate 10 μ l that draw, injection liquid chromatography, records peak area, measures once every 2 hours later, investigates 6 hours, calculates the relative standard deviation of peak area.Result shows: in test sample, in 6 hours, measurement result is stable.In Table 6, table 7.
Table 6 sample stability Bergenin test findings
Table 7 sample stability glycyrrhizic acid test findings
8. replica test
Get this product, replication 6 times, content in calculation sample.Show that analytical approach repeatability is good.
Table 8 Bergenin replica test result
Table 9 glycyrrhizic acid replica test result
9. recovery test
Precision takes 6 parts of same batch samples, and precision adds reference substance, measures its content, calculate recovery rate.Show that this assay method measurement result is accurate.
Table 10 Bergenin recovery experimental result
Table 11 glycyrrhizic acid recovery experimental result
10. sample determination
Get three batches of the peaceful particles of peace youngster, measure and calculate purple bergenia herb glycosides and glycyrrhizic acid content, result is as follows.
Table 12 sample size measurement result
| Lot number | Determination of Bergenin (mg/g) | Glycyrrhizic acid content (mg/g) |
| 20121110 | 0.71 | 0.36 |
| 20121114 | 0.72 | 0.35 |
| 20121119 | 0.83 | 0.42 |
Accompanying drawing explanation:
Fig. 1 is that Bergenin and glycyrrhizic acid mix contrast high-efficient liquid phase chromatogram;
Fig. 2 is the peaceful particle solution high-efficient liquid phase chromatogram of peace youngster;
Fig. 3 is Bergenin linear relationship chart;
Fig. 4 is glycyrrhizic acid linear relationship chart;
The horizontal ordinate of Fig. 1, Fig. 2 is the time, unit: minute (Minutes); Ordinate is voltage, unit: volt (Volts).
Following embodiment all can realize the effect described in above-mentioned experimental example.
Embodiment
Below in conjunction with embodiment, the present invention is done to detailed elaboration, but be not limited to these concrete embodiment recording.The peaceful particle of peace youngster detecting, peace youngster's Yiganning capsule Wei Jinhe Tibetan medicine Co., Ltd. Co., Ltd produce and sell.
Embodiment 1.
Purple bergenia herb, Radix Glycyrrhizae content assaying method in the peaceful particle of peace youngster, step is as follows:
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, and the volume parts of take is Mobile phase B than 0.05% phosphoric acid solution, adopts gradient elution mode: 0min → 20min → 30min → 60min, acetonitrile: 7% → 7% → 36% → 36%; Volume parts is than 0.05% phosphoric acid solution: 93% → 93% → 64% → 64%, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 3000 by glycyrrhizic acid peak;
The preparation of reference substance solution: get Bergenin reference substance, ammonium glycyrrhetate reference substance, add methyl alcohol and make every 1ml containing the solution of Bergenin, each 50 μ g of ammonium glycyrrhetate, obtain; (glycyrrhizic acid weight=ammonium glycyrrhetate weight/1.0207)
The preparation of need testing solution: will pacify after the peaceful particle porphyrize of youngster, get powder 4g, accurately weighed, put in the conical flask of tool plug, add methyl alcohol 25ml, close plug, ultrasonic processing 30min, lets cool, and supplies the weight of less loss with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, obtains;
Determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In the peaceful particle of peace youngster, the content of purple bergenia herb is in Bergenin glycosides 0.79mg/g; Radix Glycyrrhizae content is in glycyrrhizic acid 0.39mg/g.
The raw material of the above-mentioned peaceful particle of peace youngster is composed as follows:
Tabasheer 66.7g, safflower 53.3g, calculus bovis factitius 5.3g, purple bergenia herb 53.3g, Radix Glycyrrhizae 53.3g, high mountain horseradish dish 53.3g, Lagotis brachystachya Maxim 66.7g, santal 66.7g, Tangut aconite 66.7g.
Embodiment 2.
Purple bergenia herb, Radix Glycyrrhizae content assaying method in peace youngster Yiganning capsule, step is as follows:
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, and the volume parts of take is Mobile phase B than 0.05% phosphoric acid solution, adopts gradient elution mode: 0min → 20min → 30min → 60min, acetonitrile: 7% → 7% → 36% → 36%; Volume parts is than 0.05% phosphoric acid solution: 93% → 93% → 64% → 64%, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 3000 by glycyrrhizic acid peak;
The preparation of reference substance solution: get Bergenin reference substance, ammonium glycyrrhetate reference substance, add methyl alcohol and make every 1ml containing the solution of Bergenin, each 50 μ g of ammonium glycyrrhetate, obtain; (glycyrrhizic acid weight=ammonium glycyrrhetate weight/1.0207)
The preparation of need testing solution: will pacify after youngster's Yiganning capsule content porphyrize, get powder 4g, accurately weighed, put in the conical flask of tool plug, add methyl alcohol 25ml, close plug, ultrasonic processing 30min, lets cool, and supplies the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, obtain;
Determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In peace youngster Yiganning capsule, the content of purple bergenia herb is in Bergenin glycosides 0.76mg/g; Radix Glycyrrhizae content is in glycyrrhizic acid 0.42mg/g.
The raw material of above-mentioned peace youngster's Yiganning capsule is composed as follows:
Tabasheer 66.7g, safflower 53.3g, calculus bovis factitius 5.3g, purple bergenia herb 53.3g, Radix Glycyrrhizae 53.3g, high mountain horseradish dish 53.3g, Lagotis brachystachya Maxim 66.7g, santal 66.7g, Tangut aconite 66.7g.
Claims (3)
1. purple bergenia herb, a Radix Glycyrrhizae content assaying method in the peaceful preparation of Chinese medicine composition peace youngster, comprise and adopt appendix VI D high performance liquid chromatography of version < < Chinese Pharmacopoeia > > in 2010 to measure; It is characterized in that step is as follows:
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, and the volume parts of take is Mobile phase B than 0.05% phosphoric acid solution, adopts gradient elution mode: 0min → 20min → 30min → 60min, acetonitrile: 7% → 7% → 36% → 36%; Volume parts is than 0.05% phosphoric acid solution: 93% → 93% → 64% → 64%, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 3000 by glycyrrhizic acid peak;
The preparation of reference substance solution: get Bergenin reference substance, ammonium glycyrrhetate reference substance, add methyl alcohol and make every 1ml containing the solution of Bergenin, each 50 μ g of ammonium glycyrrhetate, obtain;
The preparation of need testing solution: will pacify after the peaceful preparation porphyrize of youngster, get powder 4g, accurately weighed, put in the conical flask of tool plug, add methyl alcohol 25ml, close plug, ultrasonic processing 30min, lets cool, and supplies the weight of less loss with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, obtains;
Determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains.
2. a kind of Chinese medicine composition claimed in claim 1 is pacified purple bergenia herb, Radix Glycyrrhizae content assaying method in the peaceful preparation of youngster; it is characterized in that; the described peaceful preparation of Chinese medicine composition peace youngster refers to contain purple bergenia herb and Radix Glycyrrhizae is main ingredient; contain multiple or whole in Tabasheer, safflower, calculus bovis factitius, high mountain horseradish dish, Lagotis brachystachya Maxim, santal or Tangut aconite simultaneously; technique routinely; add conventional auxiliary material to be prepared into clinical acceptable any formulation, comprise capsule, pill, micropill, dripping pill, tablet, capsule, particle, medicine materical crude slice or dispersing tablet.
3. a kind of Chinese medicine composition claimed in claim 1 is pacified purple bergenia herb in the peaceful preparation of youngster, Radix Glycyrrhizae content assaying method, it is characterized in that, a kind of bulk drug consists of Tabasheer 66.7g, safflower 53.3g, calculus bovis factitius 5.3g, purple bergenia herb 53.3g, Radix Glycyrrhizae 53.3g, high mountain horseradish dish 53.3g, Lagotis brachystachya Maxim 66.7g, santal 66.7g, purple bergenia herb in the peaceful particle of Chinese medicine composition peace youngster of Tangut aconite 66.7g, Radix Glycyrrhizae content assaying method, comprise and adopt appendix VI D high performance liquid chromatography of version < < Chinese Pharmacopoeia > > in 2010 to measure, step is as follows:
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, and the volume parts of take is Mobile phase B than 0.05% phosphoric acid solution, adopts gradient elution mode: 0min → 20min → 30min → 60min, acetonitrile: 7% → 7% → 36% → 36%; Volume parts is than 0.05% phosphoric acid solution: 93% → 93% → 64% → 64%, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 3000 by glycyrrhizic acid peak;
The preparation of reference substance solution: get Bergenin reference substance, ammonium glycyrrhetate reference substance, add methyl alcohol and make every 1ml containing the solution of Bergenin, each 50 μ g of ammonium glycyrrhetate, obtain;
The preparation of need testing solution: will pacify after the peaceful particle porphyrize of youngster, get powder 4g, accurately weighed, put in the conical flask of tool plug, add methyl alcohol 25ml, close plug, ultrasonic processing 30min, lets cool, and supplies the weight of less loss with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, obtains;
Determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In the peaceful particle of peace youngster, the content of purple bergenia herb, in Bergenin glycosides, must not be less than 0.7mg/g; Radix Glycyrrhizae content, in glycyrrhizic acid, must not be less than 0.3mg/g.
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Application publication date: 20141029 |
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