CN108008023A - A kind of detection method of brain-tonifying and kidney-nourishing Chinese medicine composition - Google Patents
A kind of detection method of brain-tonifying and kidney-nourishing Chinese medicine composition Download PDFInfo
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- CN108008023A CN108008023A CN201610976558.2A CN201610976558A CN108008023A CN 108008023 A CN108008023 A CN 108008023A CN 201610976558 A CN201610976558 A CN 201610976558A CN 108008023 A CN108008023 A CN 108008023A
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Abstract
The invention discloses a kind of detection method of brain-tonifying and kidney-nourishing Chinese medicine composition, belong to the technical field of Chinese medicine preparation quality testing.The present invention uses high performance liquid chromatography, and under same chromatographic condition, using Paeoniflorin as reference, feature qualitative analysis is carried out to contained chlorogenic acid, spinosin, forsythin, arctiin in sample.This detection method is easy, quick, sensitivity is good, stability is high, forms composition characteristic spectrum, reaches complete detection composition contained substance purpose.
Description
Technical field
The present invention relates to the field of Chinese medicines, and the quality determining method of brain-tonifying and kidney-nourishing Chinese medicine composition, particularly belongs to Chinese medicine preparation
The technical field of quality testing.
Background technology
Brain-invigorating and kidney-tonifying pill is an old kind of Chinese patent drug from the fifties in last century with regard to public offering, now record in《China
Pharmacopeia》One the 1370-1371 pages of version in 2015, full side are made of the 25 taste Chinese medicine such as red ginseng, pilose antler, dog testes and penis, have：Brain tonic is mended
Kidney, replenishes qi to invigorate the spleen, the function of tranquilizing the mind.For the forgetful insomnia caused by speen-kidney dificiency, have a dizzy spell, tinnitus, palpitaition, waist and knee
Aching and limp, seminal emission；Neurasthenia and sex dysfunction are shown in above-mentioned symptom person.
Since this product is the big prescription of 25 taste medicines, complicated.At present《Chinese Pharmacopoeia》Quality control project also only relate to
And the thin layer of a few taste medicinal materials differentiates and the assay of single Paeoniflorin, experimental implementation is cumbersome and cannot reach comprehensive control, holds
Easily occur adding single Paeoniflorin component fraud problem.Chinese medicine chromatographic characteristics collection of illustrative plates is a kind of synthesis, quantifiable Chinese medicine
And the quality detection technology of Chinese medicine preparation, it is mainly used for discerning the false from the genuine and evaluates the homogeneous of crude drug, semi-finished product and the quality of the pharmaceutical preparations
Property and stability.With the raising of automation equipment degree and the fast development of computer, substantial amounts of Chinese medicine chromatographic characteristics collection of illustrative plates
Emerge in large numbers in succession, it is current detection technique development trend to carry out related material detection with characteristic spectrum.
Since pharmaceutical compositions are complicated, during any single active ingredient cannot reflect medical drugs embodied it is whole
Physical exercise therapy is imitated, especially compound preparation, is to play curative effect by the mutual synergistic effect between multicomponent, single measure one or
Several index components cannot reflect the inherent quality of Chinese medicine or Chinese medicine compound prescription comprehensively, and Chinese medicine characteristic spectrum is a kind of comprehensive
Reflection Chinese medicine or the analysis method of chemical information that is included of Chinese medicine compound prescription.
The content of the invention
For current brain-tonifying and kidney-nourishing Chinese medicine composition quality detection technology it is simple, cannot reflect group contained by Chinese medicine compound prescription comprehensively
The problem of being divided into point, it is an object of the invention to establish brain-tonifying and kidney-nourishing Chinese medicine composition HPLC characteristic spectrum detection techniques, to it
Medicine components are effectively detected.
To achieve the above object, the present invention provides following technical solution：
The present invention uses high performance liquid chromatography, under same chromatographic condition, using Paeoniflorin as reference, to contained green original in sample
Acid, spinosin, forsythin, arctiin carry out qualitative analysis.
Wherein, characteristic spectrum detection technique method is established to carry out in the steps below：
(1) chromatographic condition：Chromatographic column is C18Column；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as Mobile phase B；Detect ripple
A length of 230nm；Flow velocity is 1ml/min, and column temperature is room temperature.
（2）The preparation of reference substance solution：Precision weigh chlorogenic acid reference substance, Paeoniflorin reference substance, spinosin reference substance,
Arctiin reference substance, forsythin reference substance are appropriate, add acetonitrile：0.1% phosphoric acid（10：90）Mixed solution is made every 1ml and respectively contains 50 μ g
Mixed solution, shake up, to obtain the final product.
（3）The preparation of test solution：Precision weighs brain-tonifying and kidney-nourishing Chinese medicine composition powder 2g, puts in conical flask with stopper,
Precision adds acetonitrile：0.1% phosphoric acid（10：90）Mixed solution 25ml, is ultrasonically treated（Power 250W, frequency 100Hz）30min, filter
Cross, take subsequent filtrate, to obtain the final product.
（4）It is accurate respectively to draw reference substance solution, 20 μ l of test solution, inject liquid chromatograph, measure, to obtain the final product.
Wherein, the step（1）Chromatographic condition and system suitability experiment：Wear peace high performance liquid chromatograph in the U.S.
（U3000 is pumped, UVD170U UV detector, CHROMELEON data processing softwares, manual injector, column oven）；Chromatographic column：
Kromasil-C18Chromatographic column（250×4.6mm）；Startorius BP211D electronic analytical balances（d=0.01mg）Vacuum outgas
System.
The brain-tonifying and kidney-nourishing Chinese medicine composition sample of 18 batches；Chlorogenic acid, Paeoniflorin, spinosin, arctiin, forsythin
Reference substance, acetonitrile are chromatographic grade, and phosphoric acid is analysis level, and water is purified water.
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as mobile phase
B, gradient elution is carried out by the regulation in table 1；Detection wavelength is 230nm.Number of theoretical plate is calculated by Paeoniflorin peak to be not less than
10000。
Table 1
| Time (minute) | Mobile phase A (%） | Mobile phase B (%) |
| 0—80 | 5→30 | 95→70 |
| 80—110 | 30→100 | 70→0 |
| 110—115 | 100 | 0 |
| 115—120 | 100→5 | 0→95 |
Access batch brain-tonifying and kidney-nourishing Chinese medicine composition sample respectively, is prepared as described above test solution, measures and record 120min
Chromatogram, as a result all chromatographic peaks concentrate in 90min, obtained 5 characteristic peaks because respectively with corresponding object of reference retain
Time is corresponding, using the corresponding peak in Paeoniflorin object of reference peak as S peaks, calculates the relative retention time of characteristic peak, its opposite reservation
Time should be within ± the 5% of setting.Setting is peak 1（Chlorogenic acid 65.36%）, peak 2（Paeoniflorin 100.00%）, peak 3
（Spinosin 122.15%）, peak 4（Forsythin 187.05%）, peak 5（Arctiin 193.65%）.
Compared with prior art, the present invention has the following advantages：
1. detection technique is advanced, the characteristic component of brain-tonifying and kidney-nourishing Chinese medicine composition can be effectively detected, and can effectively determine correlation
The corresponding structure of matter in peak, this quality of energy effective monitoring.
2. the technology of the present invention is easy to operate, detection feature is comprehensive, and reproducible, stability is good.
Brief description of the drawings
Fig. 1 is chromatogram of the mixed reference substance solution in 0~120min.
Fig. 2 is chromatogram of the brain-tonifying and kidney-nourishing Chinese medicine composition sample in 0~120min.
Embodiment
Be set forth below embodiment be further described the present invention, the embodiment be merely to illustrate the present invention and to the present invention
It is not restricted.
First, chromatographic condition and system suitability
Instrument：Wear peace high performance liquid chromatograph in the U.S.（U3000 is pumped, UVD170U UV detector, CHROMELEON data processings
Software, manual injector, column oven）；
Chromatographic column：Kromasil-C18 chromatographic columns（250×4.6mm）；
Balance：Startorius BP211D electronic analytical balances（d=0.01mg）；
Vacuum degassing system；
Chinese Pharmacopoeia Commission's Chinese medicine chromatographic characteristics collection of illustrative plates similarity evaluation system；
Mobile phase：Using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as Mobile phase B, gradient elution is carried out by the regulation in table 1；
Detection wavelength：230nm；
1 eluent gradient of table elutes table
| Time (minute) | Mobile phase A (%） | Mobile phase B (%) |
| 0—80 | 5→30 | 95→70 |
| 80—110 | 30→100 | 70→0 |
| 110—115 | 100 | 0 |
| 115—120 | 100→5 | 0→95 |
The preparation of reference substance solution：Precision weighs chlorogenic acid reference substance, Paeoniflorin reference substance, spinosin reference substance, arctiin
Reference substance, forsythin reference substance are appropriate, add acetonitrile：0.1% phosphoric acid（10：90）The mixing that every 1ml respectively contains 50 μ g is made in mixed solution
Solution, shakes up, to obtain the final product.
The preparation of test solution：Precision weighs brain-tonifying and kidney-nourishing Chinese medicine composition powder 2g, puts in conical flask with stopper, accurate
Add acetonitrile：0.1% phosphoric acid（10：90）Mixed solution 25ml, is ultrasonically treated（Power 250W, frequency 100Hz）30min, filtration,
Take subsequent filtrate, to obtain the final product.
Determination method：It is accurate respectively to draw reference substance solution, 20 μ l of test solution, liquid chromatograph is injected, is measured, i.e.,
.
There should be 5 characteristic peaks in test sample feature chromatography（See figure -1, figure -2）, should retain respectively with corresponding object of reference peak
Time is corresponding, with peak 2（Paeoniflorin）For S peaks, calculate the relative retention time of each characteristic peak and S peaks, should setting ±
Within 5%.Respectively peak 1（Chlorogenic acid 65.36%）, peak 2（Paeoniflorin 100.00%）, peak 3（Spinosin 122.15%）, peak 4
（Forsythin 187.05%）, peak 5（Arctiin 193.65%）.
2nd, methodological study
1st, precision test：Take 141203 batches of test solutions at the conditions of the experiments described above, continuous sample introduction 5 times, is investigated each shared
The relative retention time at peak and the RSD% of relative peak area.The result shows that the relative retention time RSD% of each chromatographic peak is small
In 0.25%.
2nd, stability test：Take 141203 batches of test solutions at the conditions of the experiments described above, 0h, 2h, 4h, 8h, 12h,
24h is measured, and investigates the relative retention time at each shared peak and the RSD% of relative peak area.The result shows that each chromatographic peak is opposite
Retention time is respectively less than 0.39%.
3rd, repetitive test：141203 batches of 5 parts of samples are taken, it is molten to prepare 5 parts of test samples according to test solution preparation method
Liquid, is measured at the conditions of the experiments described above, investigates the relative retention time at each shared peak and the RSD% of relative peak area.As a result
Show, the relative retention time of each chromatographic peak is respectively less than 0.28%.
3rd, sample measures：
18 batches of brain-tonifying and kidney-nourishing Chinese medicine compositions are taken, prepare 18 parts of test solutions, 20 μ of each accurate absorption respectively according to the method described above
L, is measured at the conditions of the experiments described above, records 0~120min chromatograms, when calculating opposite retain as reference peak using Paeoniflorin
Between, the characteristic spectrum of brain-invigorating Chinese medicine composite is shown in Fig. 2.
4th, the foundation of characteristic spectrum：18 batches of brain-tonifying and kidney-nourishing Chinese medicine composition samples are taken respectively, prepare test solution,
It is measured under above-mentioned experimental condition and records 120min chromatograms, as a result all chromatographic peaks is concentrated in 90min, compare 18
The chromatogram of sample, it is thus identified that 5 characteristic peaks, by with compareing collection of illustrative plates contrast, confirming peak 1（Chlorogenic acid 65.36%）, peak 2（Chinese herbaceous peony
Medicine glycosides 100.00%）, peak 3（Spinosin 122.15%）, peak 4（Forsythin 187.05%）, peak 5（Arctiin 193.65%）,
Because Paeoniflorin is the index that is detected in primary standard, therefore it is interior reference peak to select Paeoniflorin.
5th, the checking test of multiple batches of sample：
Chromatographic condition and method prepare each batch sample, measure respectively according to above-mentioned test method,.Calculate relative retention time,
The results are shown in Table 2：
The multiple batches of sample confirmatory experiment of table 2
Pass through data above：Obtained 5 characteristic peaks of each sample with corresponding object of reference because protecting respectively in 18 batches of samples
Stay the time corresponding, using the corresponding peak in Paeoniflorin object of reference peak as S peaks, calculate the relative retention time of characteristic peak, its opposite guarantor
Stay the time should be within ± the 5% of setting.Setting is peak 1（Chlorogenic acid 65.36%）, peak 2（Paeoniflorin 100.00%）, peak 3
（Spinosin 122.15%）, peak 4（Forsythin 187.05%）, peak 5（Arctiin 193.65%）.
Claims (4)
- A kind of 1. detection method of brain-tonifying and kidney-nourishing Chinese medicine composition, it is characterised in that：Using high performance liquid chromatography, chromatographic column is C18Column；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as Mobile phase B；Detection wavelength is under the chromatographic condition of 230nm, is pressed The elution program of table carries out gradient elution, using Paeoniflorin as reference, to composition Content of Chlorogenic Acid, spinosin, forsythin, burdock Glycosides etc. carries out feature qualitative analysis.
- A kind of 2. detection method of brain-tonifying and kidney-nourishing Chinese medicine composition according to claim 1, it is characterised in that：The component Content assaying method carries out in the steps below：(1) preparation of reference substance solution：Take chlorogenic acid, Paeoniflorin, spinosin, arctiin, forsythin reference substance appropriate, essence It is close weighed, add acetonitrile：Reference substance solution is made in the mixed liquor of 0.1% phosphoric acid；(2) preparation of test solution：Brain-tonifying and kidney-nourishing Chinese medicine composition powder 2g is taken, it is accurately weighed, put in conical flask with stopper, Add acetonitrile：The mixed liquor 25ml of 0.1% phosphoric acid, is ultrasonically treated, and filtration, takes subsequent filtrate, to obtain the final product；(3) determination method：It is accurate respectively to draw reference substance solution, 20 μ l of test solution, liquid chromatograph is injected, is measured, i.e., .
- A kind of 3. detection method of brain-tonifying and kidney-nourishing Chinese medicine composition according to claim 2, it is characterised in that：The step Suddenly（1）The preparation of reference substance solution：Precision weighs chlorogenic acid reference substance, Paeoniflorin reference substance, spinosin reference substance, arctiin reference substance, forsythin control Appropriate product, add 10% acetonitrile and 90%0.1% phosphoric acid mixed solution that the mixed solution that every 1ml respectively contains 50 μ g is made, shake up, to obtain the final product.
- A kind of 4. detection method of brain-tonifying and kidney-nourishing Chinese medicine composition according to claim 2, it is characterised in that：The step Suddenly（2）The preparation of test solution：Precision weighs brain-tonifying and kidney-nourishing Chinese medicine composition powder 2g, puts in conical flask with stopper, and precision adds 10% acetonitrile and 90%0.1% Phosphoric acid mixed solution 25ml, in power 250W, frequency 100Hz is ultrasonically treated 30min, and filtration, takes subsequent filtrate, to obtain the final product.
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| CN110857939A (en) * | 2018-08-23 | 2020-03-03 | 完美（中国）有限公司 | Detection method of traditional Chinese medicine composition with sleep improvement function |
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| CN101532994A (en) * | 2009-04-18 | 2009-09-16 | 姚俊华 | A method for measuring the content of a brain-invigorating and kidney-tonifying preparation |
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Cited By (3)
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| CN110857939A (en) * | 2018-08-23 | 2020-03-03 | 完美（中国）有限公司 | Detection method of traditional Chinese medicine composition with sleep improvement function |
| CN110857939B (en) * | 2018-08-23 | 2022-03-18 | 完美（中国）有限公司 | Detection method of traditional Chinese medicine composition with sleep improvement function |
| CN109030702A (en) * | 2018-11-03 | 2018-12-18 | 青海瑞成药业(集团)有限公司 | A kind of quality determining method that blood stasis-eliminating and stagnation-dissipating enema is new |
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