CN108226336A - A kind of detection method of the significant ingredient of soft capsule - Google Patents
A kind of detection method of the significant ingredient of soft capsule Download PDFInfo
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- CN108226336A CN108226336A CN201711457604.9A CN201711457604A CN108226336A CN 108226336 A CN108226336 A CN 108226336A CN 201711457604 A CN201711457604 A CN 201711457604A CN 108226336 A CN108226336 A CN 108226336A
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- ginsenoside
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- detection wavelength
- pinocembrin
- galangin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
A kind of method that HPLC measures 7 kinds of significant ingredients in panax ginseng propolis soft capsule：Using octadecylsilane chemically bonded silica as filler；Mobile phase A is 0.15% phosphoric acid solution；Mobile phase B is acetonitrile；Condition of gradient elution is 0 35min, 19%B；35 55min, 19%B → 29%B；55 70min, 29%B；70 80min, 29%B → 37%B；80 110min, 37%B；Volume flow 1.0ml/min, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1Detection wavelength is 203nm, and Chrysin, Galangin Detection wavelength are 270nm, and pinocembrin Detection wavelength is 289nm, and phenethyl caffeate Detection wavelength is 329nm.Method is easy to operate, and it is accurate to measure, and specificity is strong.
Description
Technical field
The present invention relates to a kind of high efficient liquid phase analysis method, more particularly to 7 kinds of marks in a kind of panax ginseng propolis soft capsule
The high-efficient liquid phase determining method of property ingredient.
Background technology
Panax ginseng propolis soft capsule is the soft capsule made of ginseng extract, wine bee glue powder is primary raw materials, for auxiliary
It helps hypoglycemic.Significant ingredient wherein in ginseng is ginsenoside, and the significant ingredient in propolis is flavone compound.
The total saposins assay method of document report has：Vanillin assay, thin-layered chromatography, GC methods, HPLC methods, LC-MS
Method, CE methods etc..Health food evaluates the assay method of total saposins in the health food provided with technical specification (version in 2003), is
At present in health food total saposins classical assay method.But when measuring the health food containing propolis raw material with this method, by
It is influenced in by supplies such as propolis, fillers, total saposins are difficult to be extracted efficiently in product, total saponin content measurement result
Often it is not inconsistent with practical formula ratio, poor accuracy.
CN201310035767.3 disclose it is a kind of using propolis as the health food of raw material in total saposins assay method.Packet
It includes standard curve making process, sample treatment process, test solution and crosses column process, colour developing process and total saposins calculation process, standard is bent
In line production process, curve is done using the quality of absorbance value and ginsenoside, obtains standard curve, in sample treatment process,
The health food 0.3g using propolis as raw material is taken to obtain test solution, test solution is crossed in column process, using chromatographic column to test solution
Column operation was carried out, test solution is obtained and volatilizes evaporating dish, in the process that develops the color, obtain the absorbance value of test solution, total saposins
In calculation process, obtain using propolis as the health food of raw material in total saposins content.
Since colorimetric method is without chromatographic isolation, specificity is poor, if soft using determined by ultraviolet spectrophotometry panax ginseng propolis
The total saposins and general flavone content of capsule or similar products, therefore it is easy to appear false positives, it is impossible to accurate and effective control production
Quality.Therefore it is significant in panax ginseng propolis soft capsule for detecting to need to establish a kind of accurate controllable mass analysis method
Ingredient, the shortcomings that overcome the above-mentioned prior art.
Invention content
The technical problems to be solved by the invention are to overcome drawbacks described above in the prior art, using high-efficient liquid phase color
Spectrometry measures Chrysin, Galangin, pinocembrin, phenethyl caffeate, ginsenoside Re, ginsenoside Rg1, ginseng soap simultaneously
7 kinds of significant ingredients such as glycosides Rb1 provide foundation for the formulation of ginseng bee glue soft capsule quality standard.
Technical scheme is as follows：
Using HPLC methods, chromatographic condition is as follows：
Using octadecylsilane chemically bonded silica as filler (4.6mm × 250mm；5 μm, carry carbon amounts 9~11%)；Mobile phase
A is 0.15% phosphoric acid solution；Mobile phase B is acetonitrile；Condition of gradient elution is 0-35min, 19%B；35-55min, 19%B →
29%B；55-70min, 29%B；70-80min, 29%B → 37%B；80-110min, 37%B；Volume flow 1.0ml/min,
Ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1Detection wavelength is 203nm, and Chrysin, Galangin Detection wavelength are
270nm, pinocembrin Detection wavelength are 289nm, and phenethyl caffeate Detection wavelength is 329nm, 10 μ l of sample size.
The preparation of test solution：Panax ginseng propolis soft capsule 10 is taken, cuts off, content is uniformly mixed, precision weighs
1.5g is placed in conical flask with cover, and precision adds in ethyl alcohol 50ml, weighs, and ultrasonic 40min takes out, and cools, weight is supplied with ethyl alcohol,
Shake up, filter, take subsequent filtrate to get.
Mix the preparation of contrast solution：Take Chrysin, Galangin, pinocembrin, phenethyl caffeate, ginsenoside Re,
Ginsenoside Rg1, ginsenoside Rb1's reference substance are appropriate, accurately weighed, and methanol is added to dissolve, and every 1ml is made containing Chrysin, Gao Liang
Jiang Su, pinocembrin each 1mg, phenethyl caffeate 0.4mg, ginsenoside Re, ginsenoside Rg1, each 0.7mg of ginsenoside Rb1
Mixing reference substance storing solution, precision measures above-mentioned mixing reference substance stock solution, and to add the dilution of ethyl alcohol alcohol that mixing control is made molten
Liquid, wherein per 1ml containing Chrysin, Galangin, each 100 μ g of pinocembrin, 40 μ g of phenethyl caffeate, ginsenoside Re, ginseng
Saponin(e Rg1, each 70 μ g of ginsenoside Rb1.
Propolis negative control solution：Ginseng extract is taken, soft capsule content is made by soft capsule preparation method, by for trying
Propolis negative sample solution is made in product solution manufacturing method.
Ginseng negative control solution：Wine propolis is taken, soft capsule content is made by soft capsule preparation method, by test sample
Ginseng negative sample solution is made in solution manufacturing method.
Ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1Detection wavelength is 203nm, is absorbed in end.In propolis
Complicated component, under this wavelength condition, very big interference is brought to the detection of ginsenoside, needs stringent separation condition；
For in propolis Chrysin, Galangin, pinocembrin, for phenethyl caffeate, nearby also have the interference of other peaks (as schemed
Interference Peaks before the Chrysin of peak 4 shown in 3, the Interference Peaks before 5 Galangin of peak are dry behind 6 pinocembrin of peak as shown in Figure 2
Interfering with each other between peak and peak 5 and peak 6 is disturbed, the Interference Peaks of the phenethyl caffeate above and below of peak 7 shown in Fig. 4),
Under the premise of selecting multi-wavelength detection, advanced optimize chromatographic system (if selection carries the low chromatographic column of carbon amounts, carry carbon amounts 9~
11%), so as to reduce the interference at other peaks.
Testing result of the present invention shows：There are good separating degree, ginsenoside Rg between each principal component chromatographic peak1Sample size
In 0.14~1.41 μ g (R2=0.9993)；Ginsenoside Re's sample size is in 0.14~1.39 μ g (R2=0.9992)；Ginsenoside
Rb1Sample size is in 0.14~1.44 μ g (R2=0.9996)；Chrysin sample size is in 0.20~2.04 μ g (R2=0.9994)；It is high
Alpinin sample size is in 0.20~2.01 μ g (R2=0.9992)；Pinocembrin sample size is in 0.21~2.11 μ g (R2=0.9995)；
Phenethyl caffeate sample size is in 0.08~0.82 μ g (R2=0.9991) it is in good linear relationship with peak area；Each component is averaged
The rate of recovery is respectively less than 2.0% in 97.35%~100.75%, RSD；The detection method result accurately and reliably, can be used as ginseng
The content assaying method of bee glue soft capsule quality standard.
Description of the drawings
Fig. 1：Test solution, mixing contrast solution and ginseng negative control solution HPLC under the conditions of Detection wavelength 203nm
Figure；
Fig. 2：Test solution, mixing contrast solution and propolis negative control solution HPLC under the conditions of Detection wavelength 289nm
Figure；
Fig. 3：Test solution, mixing contrast solution and propolis negative control solution HPLC under the conditions of Detection wavelength 270nm
Figure；
Fig. 4：Test solution, mixing contrast solution and propolis negative control solution HPLC under the conditions of Detection wavelength 329nm
Figure；
In Fig. 1-4：Peak 1 is ginsenoside Rg1；Peak 2 is ginsenoside Re；Peak 3 is ginsenoside Rb1；Peak 4 is Chrysin；
Peak 5 is Galangin；Peak 6 is pinocembrin；Peak 7 is phenethyl caffeate.
Specific embodiment
1 specificity of embodiment
Chromatographic condition
Agilent ZORBAX Eclipse XDB-C18(4.6mm×250mm；5 μm, carry carbon amounts 10%)；Mobile phase A is
0.15% phosphoric acid solution；Mobile phase B is acetonitrile；Condition of gradient elution is 0-35min, 19%B；35-55min, 19%B → 29%
B；55-70min, 29%B；70-80min, 29%B → 37%B；80-110min, 37%B；Volume flow 1.0ml/min, ginseng
Saponin(e Re, ginsenoside Rg1, ginsenoside Rb1Detection wavelength is 203nm, and Chrysin, Galangin Detection wavelength are 270nm,
Pinocembrin Detection wavelength is 289nm, and phenethyl caffeate Detection wavelength is 329nm.
The preparation of test solution：Panax ginseng propolis soft capsule 10 is taken, cuts off, content is uniformly mixed, precision weighs
1.5g is placed in conical flask with cover, and precision adds in ethyl alcohol 50ml, weighs, and ultrasonic 40min takes out, and cools, weight is supplied with ethyl alcohol,
Shake up, filter, take subsequent filtrate to get.
Propolis negative control solution：Ginseng extract is taken, soft capsule content is made by soft capsule preparation method, by for trying
Propolis negative sample solution is made in product solution manufacturing method.
Ginseng negative control solution：Wine propolis is taken, soft capsule content is made by soft capsule preparation method, by test sample
Ginseng negative sample solution is made in solution manufacturing method.
Mix the preparation of contrast solution：Take Chrysin, Galangin, pinocembrin, phenethyl caffeate, ginsenoside Re,
Ginsenoside Rg1, ginsenoside Rb1's reference substance are appropriate, accurately weighed, and methanol is added to dissolve, and every 1ml is made containing Chrysin, Gao Liang
Jiang Su, pinocembrin each 1mg, phenethyl caffeate 0.4mg, ginsenoside Re, ginsenoside Rg1, each 0.7mg of ginsenoside Rb1
Mixing reference substance storing solution, precision measures above-mentioned mixing reference substance stock solution, and to add the dilution of ethyl alcohol alcohol that mixing control is made molten
Liquid, wherein per 1ml containing Chrysin, Galangin, each 100 μ g of pinocembrin, 40 μ g of phenethyl caffeate, ginsenoside Re, ginseng
Saponin(e Rg1, each 70 μ g of ginsenoside Rb1.Chromatogram is shown in Fig. 1~4.The result shows that negative sample is noiseless.
Embodiment 2 is linear
Precision draw the mixing reference substance storing solution 0.5 prepared by method below " embodiment 1 " item, 1.0,2.0,2.5,3.0,
4.0ml is respectively placed in 5mL measuring bottles, with ethyl alcohol constant volume, is shaken up, with storing solution together as the mixing pair of series mass concentration
According to product solution.Each 10 μ L of above-mentioned serial mixed reference substance solution are drawn respectively, inject liquid chromatograph, record chromatogram.With peak
Area (Y) carries out linear regression to concentration (X), obtains regression equation,
Ginsenoside Rg1：Y=5991.7X+111244, R2=0.9993,14.12 μ g/ml of the range of linearity~141.20 μ g/
ml；
Ginsenoside Re：Y=10125X+12378, R2=0.9992,13.92 μ g/ml of the range of linearity~139.20 μ g/
ml；
Ginsenoside Rb1：Y=3366.2X+45705, R2=0.9996,14.39 μ g/ml of the range of linearity~143.90 μ g/
ml；
Chrysin：Y=50068X+491441, R2=0.9994,20.42 μ g/ml of the range of linearity~204.20 μ g/ml；
Galangin：Y=29078X+282400, R2=0.9992,20.14 μ g/ml of the range of linearity~201.40 μ g/ml；
Pinocembrin：Y=51476X-73619, R2=0.9995,21.08 μ g/ml of the range of linearity~210.80 μ g/ml；
Phenethyl caffeate：Y=25315X+180598, R2=0.9991,8.22 μ g/ml of the range of linearity~82.16 μ g/
ml。
3 instrument precision of embodiment
Precision draws the mixed reference substance solution that linear relationship investigates intermediate mass concentration under item, continuous sample introduction 6 times, by upper
Chromatographic condition injection liquid chromatogram is stated, chromatogram is recorded, measures each test substance peak area value.As a result ginsenoside Rg1, ginseng
Saponin(e Re, ginsenoside Rb1, Chrysin, Galangin, pinocembrin, phenethyl caffeate peak area RSD (n=6) be respectively
0.30%th, 0.31%, 0.33%, 0.22%, 0.29%, 0.37%, 0.38%, show that instrument precision is good.
4 stability of solution of embodiment
Take with a test solution, setting injector temperature is 4 DEG C, respectively at 0,2,4,6,8,12, press above-mentioned color for 24 hours
Spectral condition injects liquid chromatograph, records chromatogram, measures each Component peak area to be measured.As a result test solution is in interior color for 24 hours
Spectral peak area without significant change, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Chrysin, Galangin, pinocembrin,
The RSD of phenethyl caffeate peak area is respectively 1.04%, 1.11%, 0.93%, 1.03%, 1.07%, 0.92%,
1.48%, showing test solution, internal stability is good for 24 hours under the conditions of preparation is placed on 4 DEG C.
5 repeatability of embodiment
Same batch of 6 parts of sample is taken, by legal system available test sample solution below " embodiment 1 " item, measures containing for each substance in accordance with the law
Amount.As a result ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Chrysin, Galangin, pinocembrin, phenethyl caffeate
Average mass fraction be respectively 0.22%, 0.31%, 0.51%, 0.94%, 0.49%, 0.32%, 0.55%, RSD difference
It is 1.37%, 1.31%, 1.19%, 1.25%, 1.06%, 1.14%, 1.65%.
6 rate of recovery of embodiment
6 parts of the sample taken, every part of about 1.0g is accurately weighed, and the accurate reference substance solution that adds in is appropriate respectively, by " embodiment
Legal system available test product below 1 " item injects liquid chromatograph according to above-mentioned chromatographic condition, records chromatogram, calculates the rate of recovery.As a result
Ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Chrysin, Galangin, pinocembrin, phenethyl caffeate are averaged
The rate of recovery is 97.35%, 98.47%, 99.26%, 100.75%, 100.69%, 99.34%, 98.17%.
7 assay of embodiment
3 batches of different batches panax ginseng propolis soft capsules are taken, by legal system available test product below " embodiment 1 " item, and shine above-mentioned color
Spectral condition injects liquid chromatogram, records chromatogram, calculates content.It the results are shown in Table 1.
1 sample measurement result of table
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (4)
1. a kind of efficient liquid phase detection method of the significant ingredient of soft capsule, which is characterized in that chromatographic column is with octadecylsilane
Bonded silica gel is filler；Mobile phase A is 0.15% phosphoric acid solution；Mobile phase B is acetonitrile；Condition of gradient elution is 0-35min,
19%B；35-55min, 19%B → 29%B；55-70min, 29%B；70-80min, 29%B → 37%B；80-110min,
37%B；Volume flow 1.0ml/min, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1Detection wavelength is 203nm, white poplar
Element, Galangin Detection wavelength are 270nm, and pinocembrin Detection wavelength is 289nm, and phenethyl caffeate Detection wavelength is 329nm.
2. efficient liquid phase detection method according to claim 1, which is characterized in that the load carbon amounts of the filler for 9~
11%.
3. efficient liquid phase detection method according to claim 2, which is characterized in that the preparation method of test solution is such as
Under：
Panax ginseng propolis soft capsule 10 is taken, cuts off, content is uniformly mixed, precision weighs 1.5g and is placed in conical flask with cover,
Precision adds in ethyl alcohol 50ml, weighs, and ultrasonic 40min takes out, and cools, supplies weight with ethyl alcohol, shake up, and filters, takes subsequent filtrate, i.e.,
.
4. efficient liquid phase detection method according to claim 3, which is characterized in that mix the preparation method of contrast solution such as
Under：Take Chrysin, Galangin, pinocembrin, phenethyl caffeate, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1 couple
It is appropriate according to product, it is accurately weighed, methanol is added to dissolve, every 1ml is made containing Chrysin, Galangin, each 1mg of pinocembrin, caffeic acid benzene
Ethyl ester 0.4mg, ginsenoside Re, ginsenoside Rg1, each 0.7mg of ginsenoside Rb1 mixing reference substance storing solution, precision amount
Above-mentioned mixing reference substance stock solution is taken to add the dilution of ethyl alcohol alcohol that mixing contrast solution is made, wherein per 1ml containing Chrysin, galangal
Element, each 100 μ g of pinocembrin, 40 μ g of phenethyl caffeate, ginsenoside Re, ginsenoside Rg1, each 70 μ g of ginsenoside Rb1.
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| CN1514242A (en) * | 2003-06-19 | 2004-07-21 | 北京中医药大学 | Quality control method of injection agent for treating apoplexia |
| CN103278575A (en) * | 2013-04-28 | 2013-09-04 | 浙江大学 | Authenticity evaluating method for populus-type propolis based on multi-index quality control |
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