CN110045039A - A kind of quality determining method of ZAOREN ANSHEN YE - Google Patents
A kind of quality determining method of ZAOREN ANSHEN YE Download PDFInfo
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- CN110045039A CN110045039A CN201910408301.0A CN201910408301A CN110045039A CN 110045039 A CN110045039 A CN 110045039A CN 201910408301 A CN201910408301 A CN 201910408301A CN 110045039 A CN110045039 A CN 110045039A
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- G—PHYSICS
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- 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
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention discloses a kind of quality determining methods of ZAOREN ANSHEN YE, the detection method is using tanshin polyphenolic acid B in spinosin in semen ziziphi spinosae in high effective liquid chromatography for measuring ZAOREN ANSHEN YE, Radix Salviae Miltiorrhizae, the content of Fructus Schisandrae alcohol first, realization is comprehensively evaluated and is controlled to ZAOREN ANSHEN YE quality, to provide comprehensive, reliable foundation for the detection of the inherent quality of ZAOREN ANSHEN YE, the stability of product quality and the safety of clinical application, validity ensure that.
Description
Technical field
The present invention discloses the technical field for being related to Chinese medicine Quality Control more particularly to a kind of quality testing side of ZAOREN ANSHEN YE
Method.
Background technique
ZAOREN ANSHEN YE is now recorded in " the Sanitation Ministry medicine standard " Traditional Chinese medicine historical preparation the 18th, standard No. WS3-B-
3429-98 has the effect of bushing nourishing the liver, tranquilize the mind and promote the intelligence, is clinically used for insomnia forgetfulness caused by heart-liver blood deficiency, neurasthenia, head
The diseases such as dizzy, headache.Semen ziziphi spinosae nourishing heart yin is reused in side, beneficial liver blood is using antitoxic heart-soothing and sedative as monarch drug in a prescription；Radix Salviae Miltiorrhizae clears away heart-fire cool blood, blood-nourishing peace
Mind；Schisandra chinensis nourishing kidney-YIN, antitoxic heart-soothing and sedative are altogether ministerial drug.Three medicines share, and have bushing nourishing the liver, the function of tranquilize the mind and promote the intelligence.
Current standard Quality Control project be TLC method identify Schisandra chinensis and Radix Salviae Miltiorrhizae in prescription (using protocatechualdehyde as index at
Point).It is fixed that the characteristics of compound Chinese patent medicine mass action, determines that quality standards in Chinese drugs research should energetically carry out Chinese patent drug multicomponent
Property quantitative research work, this is also the developing direction of current " Chinese Pharmacopoeia " standard.
Currently, there is no specific quality determining method for ZAOREN ANSHEN YE, if identified using traditional thin layer, efficiently
The methods of liquid chromatogram assay is both needed to pre-treatment and relatively long analysis time, and is only capable of measurement jujube kernel peace every time
One or both of refreshing liquid ingredient, therefore there is the problems such as slow detection speed, poor reliability.
Therefore, a kind of efficient, quick quality determining method for being exclusively used in ZAOREN ANSHEN YE how is researched and developed, becomes people urgently
Problem to be solved.
Summary of the invention
In consideration of it, the present invention provides a kind of quality determining method of ZAOREN ANSHEN YE, at least to solve existing detection
Method has the problems such as slow detection speed, poor reliability.
Technical solution provided by the invention, specifically, a kind of quality determining method of ZAOREN ANSHEN YE, the detection method are adopted
Spinosin, tanshin polyphenolic acid B and three kinds of schizandrin have in the ZAOREN ANSHEN YE described in hplc simultaneous determination
Imitate the content of ingredient.
It is preferred that the quality determining method of the ZAOREN ANSHEN YE, specifically comprises the following steps:
The preparation of test solution: taking ZAOREN ANSHEN YE to mix, and measurement shakes up rear ZAOREN ANSHEN YE and is placed in volumetric flask, adds
Methanol-water mixed solution is appropriate, and shake well makes to dissolve and be diluted to scale, and filtration takes subsequent filtrate, as test solution；
The preparation of reference substance solution: weighing spinosin, tanshin polyphenolic acid B, schizandrin reference substance, be placed in volumetric flask,
Add methanol-water mixed solution to dissolve and be diluted to scale, shakes up up to mother liquor, take the mother liquor to be placed in volumetric flask, add methanol-
Water mixed solution is diluted to scale, shakes up, filtration, as reference substance solution；
Measurement: take reference substance solution and test solution, be injected separately into high performance liquid chromatograph measurement to get；
Wherein, the corresponding chromatographic condition of the high performance liquid chromatograph are as follows: with -0.1% glacial acetic acid solution (B) of acetonitrile (A)
For mobile phase, gradient elution, Detection wavelength 250nm, flow velocity 1mL/min.
Further preferably, methanol-water mixing is molten in the preparation with the preparation of the reference substance solution of the test solution
The volumetric concentration of methanol is 70% in liquid.
Further preferably, the gradient elution in the chromatographic condition specifically: 0~16min, 16%A；16~30min,
16%A → 25%A；30~40min, 25%A；40~41min, 25%A → 48%A；41~60min, 48%A → 35%A.
Further preferably, every 1mL contains 30 μ g respectively and takes spinosin, 20 μ g tanshin polyphenolic acid Bs, 10 μ g in the reference substance solution
Schizandrin.
The quality determining method of ZAOREN ANSHEN YE provided by the invention, using high effective liquid chromatography for measuring ZAOREN ANSHEN YE
Spinosin in middle semen ziziphi spinosae, tanshin polyphenolic acid B in Radix Salviae Miltiorrhizae, Fructus Schisandrae alcohol first content, realize to ZAOREN ANSHEN YE matter
Amount is comprehensively evaluated and is controlled, to provide comprehensive, reliable foundation for the detection of ZAOREN ANSHEN YE inherent quality, ensure that
The stability of product quality and safety, the validity of clinical application.
Typically, when ZAOREN ANSHEN YE spinosin content >=100 μ g/ml, danshinolic acid b content >=80 μ g/ml, the five tastes
When sub- alcohol first content >=40 μ g/ml, belong to qualified ZAOREN ANSHEN YE.
It should be understood that above general description and following detailed description be only it is exemplary and explanatory, not
Disclosure of the invention can be limited.
Detailed description of the invention
The drawings herein are incorporated into the specification and forms part of this specification, and shows and meets implementation of the invention
Example, and be used to explain the principle of the present invention together with specification.
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, for those of ordinary skill in the art
Speech, without creative efforts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the chromatogram of mixed reference substance solution, wherein 1 is spinosin, 2 is tanshin polyphenolic acid B, 3 is schisandrol
First；
Fig. 2 is the chromatogram of test solution, wherein 1 is spinosin, 2 is tanshin polyphenolic acid B, 3 is schizandrin；
Fig. 3 is the chromatogram of acid deficient jujube kernel negative sample solution, wherein 2 is tanshin polyphenolic acid B, 3 is schizandrin；
Fig. 4 is the chromatogram for lacking Radix Salviae Miltiorrhizae negative sample solution, wherein 1 is spinosin, 3 is schizandrin；
Fig. 5 is the chromatogram for lacking Schisandra chinensis negative sample solution, wherein 1 is spinosin, 2 is tanshin polyphenolic acid B.
Specific embodiment
The present invention is further expalined below with reference to specific embodiment, but be not intended to restrict the invention
Protection scope.
1, instrument and reagent
Shimadzu 20A high performance liquid chromatograph, by LC-20AT quaternary pump, SPD-M20A diode array detector (DAD),
SIL-20A autosampler and CTO-20A column oven composition.Reference substance spinosin (lot number 111869-201704), danshinolic acid
B (lot number 111562-201716), schizandrin (lot number 110857-201714) are purchased from Chinese food drug assay research
Institute.Methanol, acetonitrile are chromatographically pure, and water is redistilled water, other agents useful for same are that analysis is pure.
2, chromatographic condition and system suitability
Chromatographic column: Agilent Zorbax Eclipse Plus C18 (250mm × 4.6mm, 5 μm)；Mobile phase: acetonitrile
(A) -0.1% glacial acetic acid solution (B), gradient elution (0~16min, 16%A；16~30min, 16%A → 25%A；30~
40min, 25%A；40~41min, 25%A → 48%A；41~60min, 48%A → 35%A；；Flow velocity 1.0mLmin-1；
Detection wavelength: 250nm；Sample volume: 10 μ L.Number of theoretical plate is calculated by spinosin should be not less than 5000, the separation of each chromatographic peak
Degree is all larger than 1.5.
3, the preparation of mixed reference substance solution
It takes the reference substance of spinosin, tanshin polyphenolic acid B, schizandrin appropriate, adds 70% methanol solution that every 1mL is respectively prepared
Reference substance stock solution containing 0.3mg, 0.2mg, 0.1mg.Spinosin, tanshin polyphenolic acid B, the storage of schizandrin reference substance are taken respectively
Standby solution 10ml sets 100ml volumetric flask, adds 70% methanol solution every 1mL is made to contain 30 μ g respectively and takes spinosin, 20 μ g pellet phenol
The mixed reference substance solution of sour B, 10 μ g schizandrins.
4, the preparation of test solution
ZAOREN ANSHEN YE 10 mixings are taken, precision measures 10ml, sets in 50ml measuring bottle, add 70% methanol appropriate, sufficiently shakes
Shaking makes to dissolve and be diluted to scale, filtration, take subsequent filtrate to get.
5, negative sample solution and specificity test
Acid deficient jujube kernel is prepared respectively in prescription ratio, lacks Radix Salviae Miltiorrhizae, lacks the negative sample solution of Schisandra chinensis.By " test sample is molten
Below liquid " item method prepare solution to get.
Acid deficient jujube kernel is prepared in prescription ratio and ZAOREN ANSHEN YE preparation process, lacks Radix Salviae Miltiorrhizae, lacks the negative sample of Schisandra chinensis.
Precision draws reference substance solution, test solution and each 10 μ l of negative control sample solution, injects liquid chromatograph.The control
Product solution, test solution, the negative sample solution of acid deficient jujube kernel, the negative sample solution of scarce Radix Salviae Miltiorrhizae and the yin of scarce Schisandra chinensis
Property sample solution detection spectrogram see FIG. 1 to FIG. 5, under above-mentioned chromatographic condition, other compositions are to spinosin, red phenol in sample
Sour B, the measurement of schizandrin is noiseless.
6, linear relationship is investigated
It takes the reference substance of spinosin, tanshin polyphenolic acid B, schizandrin appropriate, adds 70% methanol solution that every 1mL is respectively prepared
Reference substance stock solution containing 0.2877mg, 0.1942mg, 0.0986mg.Spinosin, tanshin polyphenolic acid B, schisandrol are taken respectively
First reference substance stock solution 10ml sets 100ml volumetric flask, adds 70% methanol solution that every 1mL is made and contains 28.77 μ g, 19.42 μ respectively
G, the mixed reference substance solution I of 9.86 μ g；Spinosin, tanshin polyphenolic acid B, schizandrin reference substance stock solution 15ml are taken respectively
100ml volumetric flask is set, adds 70% methanol solution that the mixing control that every 1mL contains 43.16 μ g, 29.13 μ g, 14.79 μ g respectively is made
Product solution II；It takes spinosin, tanshin polyphenolic acid B, schizandrin reference substance stock solution 5ml to set 100ml volumetric flask respectively, adds
The mixed reference substance solution III that every 1mL contains 14.38 μ g, 9.71 μ g, 4.93 μ g respectively is made in 70% methanol solution.
It is accurate respectively to draw 1 μ L, 2 μ L, 5 μ L, 10 μ L, 15 μ L mixed reference substance solutions I, 15 μ L, 20 μ L mixing reference substances
Solution II, successively by above-mentioned liquid phase chromatogram condition sample introduction is analyzed measurement.The result shows that: spinosin, tanshin polyphenolic acid B, schisandrol
The sample volume of first is respectively in 0.02877~0.8631 μ g, 0.01942~0.5827 μ g, in 0.00986~0.2958 μ g range with
Peak area is in good linear relationship, and regression equation is respectively as follows: Y=1.205 × 103X+113.4, r=0.9999；Y=1.080 ×
103X-9.129×103, r=0.9992；Y=1.870 × 103X-245.2, r=0.9999.
The 1 spinosin range of linearity of table investigates result
The 2. tanshin polyphenolic acid B range of linearity of table investigates result
The 3. schizandrin range of linearity of table investigates result
7, stability test
Precision draw lot number 20190301 test solution, 0,3,6,9,15,20h distinguish 10 μ L of sample introduction, as a result this
Pi Nuosu, tanshin polyphenolic acid B, schizandrin peak area RSD be respectively 0.12%, 0.30%, 0.30%.Measurement result is shown in Table 4.
Result is investigated in 4. stability test of table
Test result shows: measuring in 20 hours after test liquid preparation, RSD value is less than 2.0%, this skin in test solution
Promise element, tanshin polyphenolic acid B, schizandrin content are basicly stable.
8, precision test
The sample of lot number 20190301 is taken, is tested by sample solution preparation method under [assay] item, precision is drawn
10 μ l of test solution injects in liquid chromatograph, continuous sample introduction 6 times, measures chromatographic peak area, and measurement result is shown in Table 5.
5 Precision test result of table
The result shows that RSD value is respectively less than 2.0%, instrument has good accuracy.
9, repetitive test
6 parts of sample of lot number 20190301 are taken, are tested by sample solution preparation method under [assay] item, sample is measured
The content of spinosin, tanshin polyphenolic acid B, schizandrin, measurement result are shown in Table 6 in product.
6 test sample assay repetitive test result of table
The result shows that RSD value, less than 2.0%, this law repeatability is good.
10, accuracy test
Using sample-adding recovery experiment method, precision measure predicted content 9 parts of ZAOREN ANSHEN YE test sample (be divided into it is low,
3 groups of middle and high concentration, contains schizandrin by every group 3 parts, the 163.30 μ g/ml containing spinosin, the 114.52 μ g/ml containing tanshin polyphenolic acid B
56.86 μ g/ml), every part of accurate 5ml test sample that measures is set in 50ml volumetric flask, and mixed reference substance solution III, I, II is separately added into
Each 25mL prepares recovery test test solution by method below the preparation of test solution " 4, " item, by above-mentioned chromatographic condition
10 μ L of sample introduction respectively calculates the rate of recovery.As a result average recovery rate (n=9) difference of spinosin, tanshin polyphenolic acid B, schizandrin
95.1%, 100.8%, 95.2%；RSD is respectively 1.9%, 3.2%, 2.5%, is specifically shown in Table 7.
7 recovery test result of table
The above test measurement result shows to measure time of spinosin in ZAOREN ANSHEN YE, tanshin polyphenolic acid B, schizandrin
Yield between respectively 91.8%~97.9%, 95.2%~104.7%, 90.3%~98.4%, RSD is respectively 1.9%,
3.2%, 2.5%, the rate of recovery is good, and method is feasible.
Those skilled in the art after considering the specification and implementing the invention disclosed here, will readily occur to of the invention its
Its embodiment.This application is intended to cover any variations, uses, or adaptations of the invention, these modifications, purposes or
Person's adaptive change follows general principle of the invention and including the undocumented common knowledge in the art of the present invention
Or conventional techniques.The description and examples are only to be considered as illustrative, and true scope and spirit of the invention are by following
Claim is pointed out.
It should be understood that the invention is not limited to the contents being described above, and its model can not departed from
It encloses and carry out various modifications and change.The scope of the present invention is limited only by the attached claims.
Claims (5)
1. a kind of quality determining method of ZAOREN ANSHEN YE, which is characterized in that using described in hplc simultaneous determination
The content of three kinds of spinosin in ZAOREN ANSHEN YE, tanshin polyphenolic acid B and schizandrin effective component.
2. the quality determining method of ZAOREN ANSHEN YE according to claim 1, which is characterized in that specifically comprise the following steps:
The preparation of test solution: taking ZAOREN ANSHEN YE to mix, and measurement shakes up rear ZAOREN ANSHEN YE and is placed in volumetric flask, adds first
Alcohol-appropriate water mixed solution, shake well make to dissolve and be diluted to scale, and filtration takes subsequent filtrate, as test solution；
The preparation of reference substance solution: spinosin, tanshin polyphenolic acid B, schizandrin reference substance are weighed, is placed in volumetric flask, adds first
Alcohol-water mixed solution dissolves and is diluted to scale, shakes up up to mother liquor, the mother liquor is taken to be placed in volumetric flask, adds methanol-water mixed
It closes solution and is diluted to scale, shake up, filter, as reference substance solution；
Measurement: take reference substance solution and test solution, be injected separately into high performance liquid chromatograph measurement to get；
Wherein, the corresponding chromatographic condition of the high performance liquid chromatograph are as follows: with -0.1% glacial acetic acid solution (B) of acetonitrile (A) be stream
Dynamic phase, gradient elution, Detection wavelength 250nm, flow velocity 1mL/min.
3. the quality determining method of ZAOREN ANSHEN YE according to claim 2, which is characterized in that the system of the test solution
The volumetric concentration of methanol is 70% in methanol-water mixed solution in the preparation of the standby and described reference substance solution.
4. the quality determining method of ZAOREN ANSHEN YE according to claim 2, which is characterized in that the ladder in the chromatographic condition
Degree elution specifically: 0~16min, 16%A；16~30min, 16%A → 25%A；30~40min, 25%A；40~41min,
25%A → 48%A；41~60min, 48%A → 35%A.
5. the quality determining method of ZAOREN ANSHEN YE according to claim 2, which is characterized in that every in the reference substance solution
1mL contains 30 μ g respectively and takes spinosin, 20 μ g tanshin polyphenolic acid Bs, 10 μ g schizandrins.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113341007A (en) * | 2021-05-24 | 2021-09-03 | 北京大学 | Method for measuring contents of multiple components in whole Chinese date seed nerve-soothing capsule based on HPLC (high performance liquid chromatography) characteristic spectrum |
| CN114487182A (en) * | 2022-01-20 | 2022-05-13 | 广西壮族自治区食品药品检验所 | Content determination method of Tianwang heart tonifying preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113341007A (en) * | 2021-05-24 | 2021-09-03 | 北京大学 | Method for measuring contents of multiple components in whole Chinese date seed nerve-soothing capsule based on HPLC (high performance liquid chromatography) characteristic spectrum |
| CN113341007B (en) * | 2021-05-24 | 2023-10-10 | 北京大学 | HPLC (high Performance liquid chromatography) characteristic spectrum-based method for measuring content of all ingredients of jujube kernel nerve-soothing capsules |
| CN114487182A (en) * | 2022-01-20 | 2022-05-13 | 广西壮族自治区食品药品检验所 | Content determination method of Tianwang heart tonifying preparation |
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