CN105974014A - Method for detecting rhizome anemarrhenae by quantitative analysis of multi-components by single marker - Google Patents
Method for detecting rhizome anemarrhenae by quantitative analysis of multi-components by single marker Download PDFInfo
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- CN105974014A CN105974014A CN201610294723.6A CN201610294723A CN105974014A CN 105974014 A CN105974014 A CN 105974014A CN 201610294723 A CN201610294723 A CN 201610294723A CN 105974014 A CN105974014 A CN 105974014A
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- chimonin
- neomangiferin
- isomangiferin
- rhizoma anemarrhenae
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 10
- 239000003550 marker Substances 0.000 title abstract 3
- 238000012360 testing method Methods 0.000 claims abstract description 43
- 230000014759 maintenance of location Effects 0.000 claims abstract description 33
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 5
- 239000009633 chimonin Substances 0.000 claims description 68
- VUWOVGXVRYBSGI-IRXABLMPSA-N 1,3,6-trihydroxy-2-[(2s,3r,4r,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]-7-[(2s,3r,4r,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyxanthen-9-one Chemical group O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(C(C2=C(O)C([C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)=C(O)C=C2O2)=O)=C2C=C1O VUWOVGXVRYBSGI-IRXABLMPSA-N 0.000 claims description 43
- VUWOVGXVRYBSGI-UHFFFAOYSA-N 7-O-beta-D-Glucopyranoside-Mangiferin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(C(C2=C(O)C(C3C(C(O)C(O)C(CO)O3)O)=C(O)C=C2O2)=O)=C2C=C1O VUWOVGXVRYBSGI-UHFFFAOYSA-N 0.000 claims description 43
- CDYBOKJASDEORM-HBVDJMOISA-N isomangiferin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(O)C2=C1OC1=CC(O)=C(O)C=C1C2=O CDYBOKJASDEORM-HBVDJMOISA-N 0.000 claims description 43
- WAYBWHJDPRHBMW-GUZANKGOSA-N neomangiferin Natural products OC[C@@H]1O[C@@H]([C@@H](O)[C@H](O)[C@H]1O)c2cc3C(=O)c4c(O)c([C@@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)c(O)cc4Oc3cc2O WAYBWHJDPRHBMW-GUZANKGOSA-N 0.000 claims description 43
- ALKWDTQJMCZSSY-UHFFFAOYSA-N isomangiferin Natural products OCC1OC(Oc2c(O)c(O)cc3C(=O)c4cc(O)c(O)cc4Oc23)C(O)C(O)C1O ALKWDTQJMCZSSY-UHFFFAOYSA-N 0.000 claims description 42
- 239000000243 solution Substances 0.000 claims description 40
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 33
- 239000012071 phase Substances 0.000 claims description 31
- 238000012937 correction Methods 0.000 claims description 26
- 239000013558 reference substance Substances 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 229930182470 glycoside Natural products 0.000 claims description 11
- 150000002338 glycosides Chemical class 0.000 claims description 11
- 239000003643 water by type Substances 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 8
- 241000605445 Anemarrhena asphodeloides Species 0.000 claims description 7
- 241001093152 Mangifera Species 0.000 claims description 7
- 239000007791 liquid phase Substances 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000012467 final product Substances 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 239000011260 aqueous acid Substances 0.000 claims 1
- 239000012488 sample solution Substances 0.000 abstract description 8
- 239000000126 substance Substances 0.000 abstract description 2
- AEDDIBAIWPIIBD-ZJKJAXBQSA-N mangiferin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(OC=2C(=CC(O)=C(O)C=2)C2=O)C2=C1O AEDDIBAIWPIIBD-ZJKJAXBQSA-N 0.000 abstract 8
- YWQSXCGKJDUYTL-UHFFFAOYSA-N Mangiferin Natural products CC(CCC=C(C)C)C1CC(C)C2C3CCC4C(C)(C)CCCC45CC35CCC12C YWQSXCGKJDUYTL-UHFFFAOYSA-N 0.000 abstract 4
- 229940043357 mangiferin Drugs 0.000 abstract 4
- 239000000523 sample Substances 0.000 description 29
- 229960000583 acetic acid Drugs 0.000 description 15
- 239000012362 glacial acetic acid Substances 0.000 description 15
- 230000008859 change Effects 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 239000003814 drug Substances 0.000 description 7
- 238000010812 external standard method Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 229930003944 flavone Natural products 0.000 description 4
- 150000002213 flavones Chemical class 0.000 description 4
- 235000011949 flavones Nutrition 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 238000003672 processing method Methods 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 241000612118 Samolus valerandi Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- 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
-
- 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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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method for detecting rhizome anemarrhenae by quantitative analysis of multi-components by a single marker. The method comprises 1, preparing a sample solution from rhizome anemarrhenae, 2, preparing a contrast solution from mangiferin, 3, respectively sucking the contrast solution and the sample solution, injecting the contrast solution and the sample solution into a high performance liquid chromatograph and carrying out a test to obtain retention time and/or content of mangiferin in the sample solution and 4, according to the retention time and/or content of mangiferin in the sample solution, carrying out qualitative analysis and/or quantitative analysis on other components in the sample solution. The method utilizes mangiferin as a contrast substance to construct quantitative analysis of rhizome anemarrhenae multi-components by a single marker so that rhizome anemarrhenae quality is well and comprehensively controlled and cost and operation problems caused by multi-component content determination are solved. The method is simple, is convenient for operation, produces accurate determination results and has good repeatability and durability.
Description
Technical field
The present invention relates to drug tests, detection method is commented in the survey particularly relating to a kind of Rhizoma Anemarrhenae more.
Background technology
The most effectively evaluating Chinese medicine quality is one of key issue of facing of Chinese medicine modern study.The many one-tenth of Chinese medicine
Point, to decide single component be Chinese medicine quality beyond expression of words to the action character of many effects, multicomponent multi objective
Control mode arise at the historic moment, and this quality controling mode brings the inspection that another problem is high
Survey expense and loaded down with trivial details detection operation.For this contradiction, a lot of experts and scholars propose new thinking, wherein
One surveys the method commented more has obtained admitting widely, and Successful utilization the containing of Multiple components in common Chinese medicine
It is fixed to measure.
The Rhizoma Anemarrhenae is the dry root of Liliaceae Anemarrhena plant Rhizoma Anemarrhenae Anemarrhenaasphodeloides Bunge
Stem, has clearing away heat-fire, promotes the production of body fluid and the effect such as moisturize.Flavones ingredient is one of main effective substance of the Rhizoma Anemarrhenae,
The most double benzene pyrrones of flavones ingredient contained in the Rhizoma Anemarrhenae, such as chimonin, Neomangiferin, Isomangiferin
Deng.Chinese Pharmacopoeia mainly uses the mensuration to chimonin, control device phase when to Rhizoma Anemarrhenae quality control
To simply, it is impossible to comprehensively control the quality of the Rhizoma Anemarrhenae.
Summary of the invention
Based on this, it is necessary to provide a survey of a kind of Rhizoma Anemarrhenae to comment detection method more.
Detection method is commented in one survey of a kind of Rhizoma Anemarrhenae more, comprises the steps:
(1) take the Rhizoma Anemarrhenae and be prepared as need testing solution；
(2) take chimonin and be configured to reference substance solution；
(3) draw described need testing solution and reference substance solution respectively, inject in high performance liquid chromatograph and carry out
Test, obtains retention time and/or the content of chimonin in described need testing solution；
(4) according to the retention time of described chimonin, remaining composition in described need testing solution is carried out qualitative point
Analysis, and/or, according to the content of described chimonin, remaining composition in described need testing solution is carried out quantitative analysis.
Wherein in an embodiment, the flowing of the employing of described high performance liquid chromatograph is mutually: with acetonitrile be
Mobile phase A, the glacial acetic acid aqueous solution of volumetric concentration 0.1-0.3% is Mobile phase B, carries out gradient elution mode:
0-8min, the percentage by volume of mobile phase A remains 9%；8-20min, the percentage by volume of mobile phase A
It is changed to 20% by 9%；20-21min, the percentage by volume of mobile phase A is changed to 9% by 20%；
21-31min, the percentage by volume of mobile phase A keeps 9%.
Wherein in an embodiment, the volumetric concentration of described glacial acetic acid aqueous solution is 0.2%.
Wherein in an embodiment, the condition of described high performance liquid chromatograph is: chromatographic column is C18 chromatograph
Post；The flow velocity of described flowing phase is 0.8-1.2mL/min；Column temperature 25-35 DEG C；Detection wavelength 256-260nm.
Wherein in an embodiment, the condition of described high performance liquid chromatograph is: chromatographic column is WatersC18 chromatographic column；The flow velocity of described flowing phase is 1.0mL/min；Column temperature 30 DEG C；Detection wavelength
258nm。
Wherein in an embodiment, remaining component described is Neomangiferin and Isomangiferin.
Wherein in an embodiment, according to step (4), the retention time of described chimonin and content, right
In described need testing solution, Neomangiferin and Isomangiferin carry out qualitative analysis and quantitative analysis:
The method of described qualitative analysis is: described Neomangiferin and Isomangiferin are relative to the phase of described chimonin
Retention time is followed successively by 0.45-0.46 and 1.08-1.09；
The retention time of described Neomangiferin or Isomangiferin is calculated by public formula (I):
Wherein, TkFor the retention time of chimonin, RtFor Neomangiferin or Isomangiferin relative to described Fructus Mangifera Indicae
The relative retention time of glycosides；
The method of described quantitative analysis is: described Neomangiferin and Isomangiferin are relative to the phase of described chimonin
Correction factor is followed successively by 0.57-0.58 and 0.9-1.0；
The content of described Neomangiferin or Isomangiferin is calculated by public formula (II):
Wherein, AkFor the peak area of chimonin, WkFor the content of chimonin, AmFor Neomangiferin or different Fructus Mangifera Indicae
The peak area of glycosides, fkmFor Neomangiferin or Isomangiferin relative to the relative correction factor of described chimonin.
Wherein in an embodiment, relative relative to described chimonin of described Neomangiferin and Isomangiferin
Retention time is followed successively by 0.46 and 1.09；
Described Neomangiferin and Isomangiferin are followed successively by 0.574 relative to the relative correction factor of described chimonin
With 0.929.
Wherein in an embodiment, the preparation method of step (1) described need testing solution is:
Accurately weighed Common Anemarrhena Rhizome, Common Anemarrhena Rhizome precision described in every 0.1g adds Diluted Alcohol 20-30mL, ultrasonic
After processing 20-40min, filter with the microporous filter membrane of 0.22-0.45 μm, take subsequent filtrate, to obtain final product.
Wherein in an embodiment, the preparation method of step (2) described reference substance solution is:
Precision weighs chimonin, and accurate addition Diluted Alcohol dissolves and is configured to 0.01-0.1mg mL-1Solution,
Obtain.
Compared with prior art, the method have the advantages that
The present invention is using chimonin as reference substance, and detection method is commented in the survey constructing the Rhizoma Anemarrhenae more, the most permissible
More preferably more fully control the quality of the Rhizoma Anemarrhenae, evade simultaneously and being asked by the cost becoming assay to bring and operation more
Topic, method is the most convenient to operate, and measurement result is accurate, and method repeatability, ruggedness is good.
Accompanying drawing explanation
Fig. 1 is blank solvent (A) HPLC figure；
Fig. 2 is mixing reference substance (B) HPLC figure；
Fig. 3 is Rhizoma Anemarrhenae sample (C) HPLC figure；
Fig. 4 is the maximum absorption spectrum figure of chimonin, Neomangiferin, Isomangiferin；
Fig. 5 is the HPLC figure that sample separates under the conditions of different wave length；
Fig. 6 is the HPLC figure that sample separates under the conditions of different chromatographic column；
Fig. 7 is the HPLC figure that sample separates under the conditions of different flowing phase pH；
Fig. 8 is the HPLC figure that sample separates under the conditions of different column temperature；
Fig. 9 is the HPLC figure that sample separates under the conditions of different in flow rate.
Detailed description of the invention
Comment detection method to make a survey of the Rhizoma Anemarrhenae of the present invention below in conjunction with specific embodiment further details of more
Explanation.
1 instrument and reagent
DIONEX Ultimate 3000 highly effective liquid phase chromatographic system；Agilent1200 series of high efficiency liquid chromatograph
System；Waters 2695 highly effective liquid phase chromatographic system；Chromatographic column WatersC18(5 μm, 250 × 4.6
mm)；Chromatographic column Agilent ZORBAX Eclipse XDB-C18(5 μm, 250 × 4.6mm)；Chromatographic column
phenomenexNX-C18 (5 μm, 250 × 4.6mm)；XS204 type electronic balance (d=0.1mg,
Mettler Toledo)；MX5 type electronic balance (d=0.001mg, Mettler Toledo)；RHP-100 type is high
Speed multifunctional crusher (Yongkang City, Zhejiang Rong Hao Trade Co., Ltd.)；DHG-9245A type electric heating air blast is done
Dry case (the permanent scientific instrument company limited in Shanghai City one)；KQ-500DA type numerical control supersonic cleaning device (40KHz
&500W, Kunshan Ultrasonic Instruments Co., Ltd.).
Chimonin (111607-200402, National Institute for Food and Drugs Control), (Chengdu is auspicious for Neomangiferin
Fen Si bio tech ltd, through area normalization method measure, purity be more than 98%), Isomangiferin (become
All Man Site bio tech ltd, measures through area normalization method, purity 99.81%)；Acetonitrile (chromatograph
Pure, Merck), glacial acetic acid (HPLC level, Aladdin), water is ultra-pure water, and other reagent are analytical pure.
The Rhizoma Anemarrhenae is to pick up from Hui nationality and the Rhizoma Anemarrhenae of Hebei Anguo, identifies being hundred through Lian Linsheng senior engineer
The dry rhizome of the conjunction section plant Rhizoma Anemarrhenae (AnemarrhenaasphodeloidesBge.).
2 methods and result
2.1 chromatographic condition
Chromatographic column Waters XBridge C18(250 × 4.6mm, 5 μm)；Flowing phase acetonitrile (A) and 0.2%
Glacial acetic acid (B), carries out gradient elution, flow velocity 1.0mL min by the regulation in table 1-1, column temperature is 30 DEG C,
Detection wavelength is 258nm, and sample size is 5 μ L.Under this chromatographic condition, each composition separating degree to be measured is good,
Blank solvent, mixing reference substance are shown in Fig. 1-3 with Rhizoma Anemarrhenae sample chromatogram figure.
Table 1 condition of gradient elution table
The preparation of 2.2 reference substance mixing storing solutions:
Take Neomangiferin, chimonin, Isomangiferin reference substance respectively in right amount, accurately weighed, add Diluted Alcohol (in "
State's pharmacopeia one " annex XVB test solution, when 20 DEG C, volumetric concentration is 49.5-50.5%) dissolve, it is configured to contain
Neomangiferin 0.2mg mL-1, chimonin 0.1mg mL-1, Isomangiferin 0.025mg mL-1Mixing comparison
Product storing solution, to obtain final product.
The preparation of chimonin reference substance solution: take chimonin reference substance appropriate, accurately weighed, add Diluted Alcohol and dissolve,
It is configured to 0.05mg mL-1List mark solution, to obtain final product.
The preparation of 2.3 need testing solutions:
Take Common Anemarrhena Rhizome (crossing No. three sieves) about 0.1g, accurately weighed, put in tool plug conical flask, accurate addition
Diluted Alcohol 25mL, weighed weight, supersound process (power 400W, frequency 40kHz) 30 minutes, let cool,
The most weighed weight, supplies the weight of less loss, shakes up with Diluted Alcohol.Filter with the micropore of 0.22 μm, take continuous
Filtrate, to obtain final product.
2.4 system suitability test
Precision draws blank solvent, mixing reference substance solution, each 5 μ L of need testing solution respectively, and sample introduction divides
Analysis, result is shown in Fig. 1.The separating degree that Neomangiferin, chimonin, Isomangiferin are adjacent chromatographic peak is the biggest
In 1.5, tailing factor Neomangiferin 1.00～1.02, chimonin 1.05～1.10, Isomangiferin 1.03～1.07,
Theoretical cam curve in terms of chimonin chromatographic peak more than 50000.
2.5 the range of linearity
Take the mixing reference substance storing solution under " 2.2 " item, accurate dilutions 1,2,4,5,10,20 times, preparation
Becoming the mixing reference substance solution of series, each concentration respectively sample introduction 5 μ L, with the sample size of each reference substance to peak
Area carries out regression treatment, obtains regression equation and the correlation coefficient of Neomangiferin, chimonin and Isomangiferin
R, result shows, the sample size of each reference substance and peak area are all in good linear relationship (table 2).
Table 2 range of linearity investigates result
2.6 correction factors and relative retention time calculate
Take the mixing reference substance solution of the series concentration of preparation under " 2.5 " item, each concentration sample introduction 5 μ L respectively.
With chimonin as internal standard, by formula(AkFor object of reference peak area, WkDense for object of reference
Degree, AmFor other component m peak areas, WmFor other component m concentration) and(TkFor object of reference
Retention time, TmFor other component m retention times), calculate chimonin respectively to Neomangiferin and different Fructus Mangifera Indicae
The correction factor of glycosides and relative retention time, average, and result shows, with chimonin as object of reference, newly
The response factor of chimonin and Isomangiferin is respectively 0.574 and 0.929, and relative retention time is respectively 0.46
With 1.09, RSD is respectively less than 2% (table 3).
Table 3 correction factor and relative retention time
2.7 precision test
The same mixing reference substance solution 5 μ L of accurate absorption, continuous sample introduction 6 times, record peak area.Precision
Investigation result shows, Neomangiferin peak area RSD is 0.60%, and chimonin peak area RSD is 0.26%,
Isomangiferin peak area RSD is 1.2%, and RSD is respectively less than 2.0%, illustrates that instrument precision is good (table 4).
Table 4 precision investigates test
2.8 stability test
Take same need testing solution, respectively at place 0,2,4,6,8,12,24,48h time sample introduction divide
Analysis, measures each determinand peak area, calculates content.Study on the stability result shows, in 0～24h, and new awns
Really glycosides content RSD is 0.45%, and chimonin content RSD is 1.3%, and Isomangiferin content RSD is
0.83%, RSD are respectively less than 2.0%, explanation process after sample solution in Neomangiferin, chimonin, different
Chimonin is relatively stable (table 5) in 24h.
Table 5 study on the stability is tested
2.9 replica test
Take with a collection of Rhizoma Anemarrhenae, by legal system available test sample solution 6 parts below " 2.3 " item, record Neomangiferin, awns
Really glycosides, the average content of Isomangiferin are respectively 1.25%, 1.10%, 0.098%, and RSD is respectively
0.82%, 0.73%, 1.3%, RSD are respectively less than 2.0%, and illustration method repeatability is good (table 6).
Table 6 repeatability investigates test
2.10 average recovery is tested
Precision weighs the Common Anemarrhena Rhizome (crossing No. 3 sieves) 6 parts of appropriate known content, and it is a certain amount of right to be separately added into
According to product, prepare sample by need testing solution preparation method under " 2.3 " item, measure content, calculate sample-adding and reclaim
Rate.Result Neomangiferin, chimonin, the average recovery of Isomangiferin be respectively 95.5%, 100.6%,
102.1%, RSD are respectively 1.9%, 1.1%, 2.5%, all meet the requirement (table of average recovery experiment
7)。
Table 7 average recovery investigates test
2.11 chromatographic condition is investigated
Prepare test sample according to the preparation method of test sample under " 2.3 " item, take the series concentration of preparation under " 2.5 " item
Mixing reference substance solution, sampling volume is 5 μ L.Investigate the chromatographic column of 3 different manufacturers respectively
(WatersC18、Agilent ZORBAX Eclipse XDB-C18、phenomenexNX-C18), column temperature changes ± 5 DEG C, detection wavelength change ± 2nm, change in flow ± 20%, with
And difference flowing phase pH value, instrument chromatographic behavior change when being measured.Relatively with new in a collection of Rhizoma Anemarrhenae
Chimonin (Neo), chimonin (M), the mensuration content of Isomangiferin (Iso) 3 compositions, and compare
Its separating degree, tailing factor, theoretical cam curve.
(1) wavelength selects
Use DAD full wavelength scanner reference substance solution, measure Neomangiferin, chimonin and Isomangiferin respectively
Maximum absorption wavelength.Ultraviolet spectrogram shows, Neomangiferin has maximum absorption wavelength at 256.5nm,
Chimonin has maximum absorption wavelength at 257.8nm, and Isomangiferin has maximum absorption wave at 255.9nm
Long.Compare the content of three compositions at 256nm, 258nm, 260nm simultaneously, no significant difference, because of
This detection wavelength is ultimately set to 258nm (table 8, Fig. 4, Fig. 5).
Table 8 different wave length experimental result (n=6)
(2) chromatographic column is investigated
Investigate WatersC18、Agilent ZORBAX Eclipse XDB-C18、phenomenexNX-C18The impact that sample is separated by the chromatographic column of three kinds of different brands.Result shows, three
Chromatographic column all can obtain good separation to target compound, meets and analyzes requirement, it is contemplated that WatersC18Peak shape preferable, tailing factor is little compared with the chromatographic column of other two brands, finally selects
WatersC18Carry out testing (table 9 and Fig. 6).
The different chromatographic column of table 9 and high performance liquid chromatograph investigate (n=6)
(3) flowing phase pH value is investigated
Investigate different pH flow visualizing (water, 0.1% glacial acetic acid, 0.2% glacial acetic acid, 0.3% glacial acetic acid) right
The impact of sample separation situation.Result shows, adds glacial acetic acid and can be obviously improved chromatographic peak inside flowing mutually
Peak shape, and compare 0.1% glacial acetic acid, 0.2% glacial acetic acid and the system of 0.3% glacial acetic acid, then peak shape is without bright
Significant difference is different, but the chimonin content difference of different pH value is bigger, it is contemplated that the acid chromatograph to target component
Behavior has stronger impact, for reaching relatively stable analytical effect, and final choice 0.2% glacial acetic acid system
Carry out testing (table 10 and Fig. 7).
Table 10 different flowing phase pH value experimental result (n=6)
(4) column temperature is investigated
Investigate the impact that sample is separated by different column temperature (25 DEG C, 30 DEG C, 35 DEG C).Result shows, three
At a temperature of separating degree no significant difference, all can reach good separating effect, when column temperature is relatively low, theoretical tower
Plate number is higher, but peak shape is poor, and the theoretical cam curve at 30 DEG C is high compared with 35 DEG C, considers the reality of method
By property, 30 DEG C of column temperatures are selected to carry out testing (table 11 and Fig. 8).
The different column temperature experimental result (n=6) of table 11
(5) flow velocity is investigated
Investigate different in flow rate (0.8mL min-1、1.0mL·min-1、2.0mL·min-1) to sample separate shadow
Ring.Result shows, separating degree no significant difference under three flow velocitys, and the theoretical cam curve under low flow velocity is higher,
And contrast the symmetrical factor of three, 1.0mL min-1There is more superior peak shape, final selection 1.0mL min-1
Carry out testing (table 12 and Fig. 9).
Table 12 different in flow rate experimental result (n=6)
2.12 serviceability test
Prepare test sample according to the preparation method of test sample under " 2.3 " item, take the series concentration of preparation under " 2.5 " item
Mixing reference substance solution, sampling volume is 5 μ L.With chimonin as internal standard, calculate Neomangiferin respectively
With the relative correction factor of Isomangiferin, average.Investigate column temperature change ± 5 DEG C respectively, detect wavelength
Change ± 2nm, change in flow ± 20%, flowing phase pH value change, and use the chromatograph of 3 different manufacturers
Post (WatersC18、Agilent ZORBAX Eclipse XDB-C18、phenomenexNX-C18), the chromatographic system of 3 different brands (Waters 2695, Agilent1200,
DIONEX Ultimate 3000) change of correction factor when being measured.Meanwhile, in order to only with awns
When really glycosides is reference substance, it is possible to confirm Neomangiferin and the position of Isomangiferin chromatographic peak, compare different condition
Under other 2 the composition chromatographic peaks to be measured relative retention time peak-to-peak with chimonin chromatograph.
(1) detector wavelength
Investigate the impact that standard solution is measured by different wave length 256nm, 258nm, 260nm, calculate correction
The factor and relative retention time, and combine corresponding system suitability parameter, result table during chromatographic condition is investigated
Bright, relative retention time and system suitability parameter are had no significant effect by wavelength change, but correction factor is big
In 2.0%, wavelength is relatively big for the impact of this correction factor, needs to control wavelength (table 13).
The different detector wavelength of table 13 investigates (n=6)
(2) chromatographic column and highly effective liquid phase chromatographic system
The different chromatographic column of investigation and chromatograph are on correction factor and the impact of relative retention time, and result shows,
The RSD of the correction factor of each composition is between 1.4%～4.0%, and the RSD of relative retention time exists
Between 1.4%～4.6%, show that the method ruggedness is preferably (table 14).
The different chromatographic column of table 14 and highly effective liquid phase chromatographic system investigate (n=6)
(3) flowing phase pH value
(0.1% glacial acetic acid aqueous solution, 0.2% glacial acetic acid are water-soluble to investigate the system of three different flowing phase pH value
Liquid, 0.3% glacial acetic acid aqueous solution) on standard solution measure impact, calculate correction factor with relative reservation time
Between, and combining corresponding system suitability parameter during chromatographic condition is investigated, result shows, flow phase pH value
The correction factor of Neomangiferin there is certain impact, in order to ensure that the reliability of result answers moderate control scope
(table 15).
Table 15 different flowing phase pH value ruggedness investigates (n=6)
(4) column temperature
Investigate the impact that standard solution is measured by different column temperature (25 DEG C, 30 DEG C, 35 DEG C), calculate correction factor
And relative retention time, and combine corresponding system suitability parameter during chromatographic condition is investigated, result shows,
Column temperature is less on impacts such as the correction factor of each composition, relative retention time, can select under normal circumstances
30 DEG C carry out testing (table 16).
Different column temperature ruggedness experimental result (n=6) of table 16
(5) flow velocity
Investigate different flow velocity (0.8mL min-1、1.0mL·min-1、2.0mL·min-1) standard solution is measured
Impact, calculate correction factor and relative retention time, and the corresponding system during chromatographic condition is investigated that combines fitted
Answering property parameter, result shows, flow velocity is less on correction factor and the relative retention time impact of each composition,
RSD is less than 2%, good tolerance (table 17).
Table 17 different in flow rate ruggedness investigates (n=6)
2.13 sample determination
Precision draws test sample, the mixing reference substance of series concentration, each 5 μ L notes of chimonin list mark solution respectively
Enter high performance liquid chromatograph, measure.Be respectively adopted a survey comments method and external standard method to calculate new Fructus Mangifera Indicae in the Rhizoma Anemarrhenae more
Glycosides, chimonin, the content of Isomangiferin, wherein uses a survey to comment the computing formula of method to be more:(AkFor object of reference peak area, WkFor object of reference concentration, AmFor other component m peak areas,
WmFor other component m concentration).Result shows, one survey comment more Neomangiferin measured by method and external standard method,
Chimonin, the content no significant difference of Isomangiferin, a survey comments method to can be used to measure the Rhizoma Anemarrhenae 3 composition more
Content (table 18).
The content (method and external standard method are commented in a survey more) of 3 kinds of flavones ingredients in the table 18 separate sources Rhizoma Anemarrhenae
2.15 with the results contrast of the mensuration of official method (chimonin content)
The test sample processing method of the present invention measures with the Rhizoma Anemarrhenae chimonin under 2015 editions " Chinese Pharmacopoeia " items
Test sample processing method is consistent, prepares basic, normal, high three concentration by test sample processing method under " 2.3 " item,
Totally 6 parts of Rhizoma Anemarrhenae need testing solutions, respectively according to the mensuration of the condition determination in the present invention He " Chinese Pharmacopoeia "
Condition measures the content of chimonin, compares the method measuring chimonin and pharmacopeia measurement result that the present invention sets up
Difference.Result shows, according to the present invention set up assay method measure chimonin content with according to medicine
The result no significant difference (table 19) that allusion quotation measures.
The results contrast of the mensuration of table 19 and official method (chimonin content)
In summary, the present invention uses HPLC method, with chimonin as reference substance, with external standard method its
Content in the Rhizoma Anemarrhenae, measures the relative correction factor of Neomangiferin, Isomangiferin, then with obtaining simultaneously
Relative correction factor calculate Neomangiferin, the content of Isomangiferin, it is achieved that one survey comment more.Use again external standard
Method determines the content of Neomangiferin in the Rhizoma Anemarrhenae, Isomangiferin, compares it and uses relative correction factor meter
Difference between calculation value.Result shows that the content value that each composition uses relative correction factor to calculate is surveyed with external standard method
There was no significant difference between definite value.One surveys and comments the suitability of method and feasibility at 3 kinds of flavones ingredients of the Rhizoma Anemarrhenae more
Assay in be verified.Meanwhile, the present invention also provide a comparison assay method (the only chimonin of pharmacopeia
Assay), result shows consistent with pharmacopeia testing result, i.e. this method while taking into account pharmacopeia index also
The quality control of other compositions can be taken into account, while not increasing cost, it is achieved that Rhizoma Anemarrhenae quality control simultaneously
The lifting of system.
Each technical characteristic of embodiment described above can combine arbitrarily, for making description succinct, the most right
The all possible combination of each technical characteristic in above-described embodiment is all described, but, if these skills
There is not contradiction in the combination of art feature, is all considered to be the scope that this specification is recorded.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed,
But can not therefore be construed as limiting the scope of the patent.It should be pointed out that, for this area
For those of ordinary skill, without departing from the inventive concept of the premise, it is also possible to make some deformation and change
Entering, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended power
Profit requires to be as the criterion.
Claims (10)
1. detection method is commented in a survey of a Rhizoma Anemarrhenae more, it is characterised in that comprise the steps:
(1) take the Rhizoma Anemarrhenae and be prepared as need testing solution；
(2) take chimonin and be configured to reference substance solution；
(3) draw described need testing solution and reference substance solution respectively, inject in high performance liquid chromatograph and carry out
Test, obtains retention time and/or the content of chimonin in described need testing solution；
(4) according to the retention time of described chimonin, remaining composition in described need testing solution is carried out qualitative point
Analysis, and/or, according to the content of described chimonin, remaining composition in described need testing solution is carried out quantitative analysis.
Detection method is commented in one survey of the Rhizoma Anemarrhenae the most according to claim 1 more, it is characterised in that described height
The flowing of the employing of effect liquid phase chromatogram instrument is mutually: with acetonitrile as mobile phase A, the ice second of volumetric concentration 0.1-0.3%
Aqueous acid is Mobile phase B, carries out gradient elution mode: 0-8min, and the percentage by volume of mobile phase A is protected
Hold is 9%；8-20min, the percentage by volume of mobile phase A is changed to 20% by 9%；20-21min, flowing
The percentage by volume of phase A is changed to 9% by 20%；21-31min, the percentage by volume of mobile phase A keeps
9%.
Detection method is commented in one survey of the Rhizoma Anemarrhenae the most according to claim 2 more, it is characterised in that described ice
The volumetric concentration of acetic acid aqueous solution is 0.2%.
Detection method is commented in one survey of the Rhizoma Anemarrhenae the most according to claim 1 more, it is characterised in that described height
The condition of effect liquid phase chromatogram instrument is: chromatographic column is C18 chromatographic column；The flow velocity of described flowing phase is
0.8-1.2mL/min；Column temperature 25-35 DEG C；Detection wavelength 256-260nm.
Detection method is commented in one survey of the Rhizoma Anemarrhenae the most according to claim 4 more, it is characterised in that described height
The condition of effect liquid phase chromatogram instrument is: chromatographic column is WatersC18 chromatographic column；Described flowing phase
Flow velocity is 1.0mL/min；Column temperature 30 DEG C；Detection wavelength 258nm.
The Rhizoma Anemarrhenae the most according to claim 1 one survey comment detection method more, it is characterised in that described its
Remaining component is Neomangiferin and Isomangiferin.
7. want a survey of the Rhizoma Anemarrhenae described in 6 to comment detection method according to right, it is characterised in that step (4) more
According to the retention time of described chimonin and content, to Neomangiferin in described need testing solution and different Fructus Mangifera Indicae
Glycosides carries out qualitative analysis and/or quantitative analysis:
The method of described qualitative analysis is: described Neomangiferin and Isomangiferin are relative to the phase of described chimonin
Retention time is followed successively by 0.45-0.46 and 1.08-1.09；
The retention time of described Neomangiferin or Isomangiferin is calculated by public formula (I):
Wherein, TkFor the retention time of chimonin, RtFor Neomangiferin or Isomangiferin relative to described Fructus Mangifera Indicae
The relative retention time of glycosides；
The method of described quantitative analysis is: described Neomangiferin and Isomangiferin are relative to the phase of described chimonin
Correction factor is followed successively by 0.57-0.58 and 0.9-1.0；
The content of described Neomangiferin or Isomangiferin is calculated by public formula (II):
Wherein, AkFor the peak area of chimonin, WkFor the content of chimonin, AmFor Neomangiferin or different Fructus Mangifera Indicae
The peak area of glycosides, fkmFor Neomangiferin or Isomangiferin relative to the relative correction factor of described chimonin.
8. want a survey of the Rhizoma Anemarrhenae described in 7 to comment detection method according to right, it is characterised in that described new awns more
Really glycosides and Isomangiferin are followed successively by 0.46 and 1.09 relative to the relative retention time of described chimonin；
Described Neomangiferin and Isomangiferin are followed successively by 0.574 relative to the relative correction factor of described chimonin
With 0.929.
9. comment detection method according to a survey of the Rhizoma Anemarrhenae described in any one of claim 1-8 more, it is characterised in that
The preparation method of step (1) described need testing solution is:
Accurately weighed Common Anemarrhena Rhizome, Common Anemarrhena Rhizome precision described in every 0.1g adds Diluted Alcohol 20-30mL, ultrasonic
After processing 20-40min, filter with the microporous filter membrane of 0.22-0.45 μm, take subsequent filtrate, to obtain final product.
10. comment detection method according to a survey of the Rhizoma Anemarrhenae described in any one of claim 1-8 more, it is characterised in that
The preparation method of step (2) described reference substance solution is:
Precision weighs chimonin, and accurate addition Diluted Alcohol dissolves and is configured to 0.01-0.1mg mL-1Solution,
Obtain.
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| CN114965747A (en) * | 2022-04-28 | 2022-08-30 | 广东一方制药有限公司 | Method for constructing fingerprint of traditional Chinese medicine formula granule by using gypsum-anemarrhena medicine and method for detecting content of index components |
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