CN106053626A - Method for rapidly measuring active components namely echinacoside and verbascoside of cistanche - Google Patents
Method for rapidly measuring active components namely echinacoside and verbascoside of cistanche Download PDFInfo
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- 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
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Abstract
The invention relates to a method for rapidly measuring the active components namely echinacoside and verbascoside of cistanche. According to the provided method, a high performance liquid chromatography-mass spectrum (HPLC-MS) technology is used to quantitatively measure the contents of two active components namely echinacoside and verbascoside of cistanche. The method is tested by experiments, has the advantages of faster analysis speed (a test can be finished within five minutes), high sensitivity, and good repeatability, and is capable of rapidly and effectively measuring two active component contents of cistanche.
Description
Technical field
The present invention relates to a kind of echinacoside and the method for verbascoside active component in quick mensuration Herba Cistanches, especially
Relate to the use of Ultra Performance Liquid Chromatography-mass spectrometric hyphenated technique and the most quickly analyze echinacoside, verbascose in mensuration Herba Cistanches
The method of glycosides active component content.
Background technology
Herba Cistanches (Cistanche deserticola Ma) is Orobanchaceae Orobanchaceae Cistanche deserticola
Cistanche Hoffmg.et Link, its medicinal effects is dry meat stem, has a kidney-replenishing, benefiting essence-blood, loosening bowel to relieve constipation it
Effect, its main active ingredient is phenethyl alcohol glycoside compounds.Two kinds of main one-tenth in existing document report detection Herba Cistanches
Point but the time of operation relatively long, the longest needs 60 minutes could by two components separately, and this method uses mass detector,
And at chromatographic column, flow velocity, the operation time is all different from the document of following report, particularly the operation time is foreshortened to 5 points
Clock.
The HPLC that appeared in the newspapers in Chinese Pharmaceutical Journal 39 (10): 740-741 in 2004 measures pine in Herba Cistanches medical material simultaneously
The really content (Zhang Siju) of echinacoside and verbascoside, YMC-Pack ODS-A used (5 μm, 25*4.6mm) chromatographic column, 30 DEG C
Column temperature, CH3CN-MeOH-1%HAc (10:15:75) is flowing phase, flow velocity 0.7mL/min, detects wavelength 334nm, runs the time
40 minutes.CHINA JOURNAL OF CHINESE MATERIA MEDICA 2005, echinacoside and verbascose glycosides in the Cistanche Tubulosa reported in 30 (11): 839-841
Assay (Chen Min), chromatographic column used is Agilent Eclipse DB-C18 (4.6*250mm, 5 μm), column temperature, flowing
Phase and detection wavelength there is no improvement, and simply flow velocity changes 0.6mL/min into, runs 25 minutes time.The Chinese pharmacy of 2007 is miscellaneous
Will Shimadzu cup whole nation pharmaceutical analysis best paper competition exchanging meeting there is a report HPLC to measure pine in CONGRONG TONGBIAN KOUFUYE
The really content (Liu Lansheng) of echinacoside and verbascoside, Hypersil C18 (4.6mm × 250mm, 5 μm)；Flowing phase: acetonitrile-
Methanol one 1% acetum (10:l0:80)；Column temperature, detection wavelength improves with the first two year document report nothing；Flow velocity changes 1mL/ into
min；The operation time is 40 minutes.
The high effective liquid chromatography for measuring compound recipe Herba Cistanches oral liquid of report in the medical science Leader 28 (2): 231-232 of 2009
Echinacoside and verbascoside content (Kang Airong), the method uses YMC-Pack ODS-A (6.0mm × 150mm, 5 μm) color
Spectrum post, 35 DEG C of column temperatures, flowing is methanol-0.2% formic acid solution mutually, gradient elution, and flow velocity brings up to 1.0mL/min, detects ripple
Long 334nm is constant, and the operation time is 30 minutes.The middle co-occurrence in the same year is surveyed for the HPLC of report in medicinal application 15:14-16 simultaneously
The content (Huo Yanxian) determining echinacoside and verbascoside in the prebiotic ball of primordial qi strengthening uses high performance liquid chromatography, with YMC-
PackODS-A (250mm × 4.6mm, 5 μm) chromatographic column is fixing phase, and acetonitrile-methanol-1% acetic acid aqueous solution (10:13:77) is
Flowing phase, column temperature is 30 DEG C, and flow velocity and detection wavelength are the most unchanged, simply operation time 45 minutes.
The high effective liquid chromatography for measuring width intestinal Liqi Decoction of report in the Chinese herbal medicine 41 (12): 1994-1995 of 2010
Middle echinacoside and verbascoside (Zhang Yan), the method uses chromatographic column to be Agilent C18 (250mm × 4.6mm, 5 μm),
Flowing carries out gradient elution for acetonitrile-1% glacial acetic acid mutually, and column temperature is increased to be 40 DEG C, and flow rate detection wavelength is not still any change,
The operation time is 35 minutes.
In Chinese patent medicine 35 (12): 2649-2653 in 2013, the HPLC method of report measures and exempts from catechin, table in numbness Kang Sheng Pill
Theine, echinacoside and verbascoside (Hou Ying), Hypersil C18 chromatographic column (4.6mm × 150mm, 5 μm), flow velocity is still
1.0mL/min, mobile phase A is l% glacial acetic acid solution, and Mobile phase B is acetonitrile-methanol (1: 4), detects wavelength 330nm, during operation
Between 8 minutes.
Chinese pharmacists, in 2014 (8): 1336-1338 the HPLC method of report measure in kidney tonifying heart tonifying capsule echinacoside and
The content (Lu Caiyang) of verbascoside, uses chromatographic column Shim-pack VP-ODS C18, and flowing is methanol-0.1% first mutually
Acid solution carries out gradient elution, and detection wavelength is that 330nm is constant, and flow velocity is with document report last year and unchanged, and column temperature is 28
℃.35 minutes operation time.During the same year in treasure's traditional Chinese medical science traditional Chinese medicines 5:1191-1193 report Cistanche Tubulosa difference growth time and
The active constituent content analysis (Yang Taixin) at position, chromatographic column Agilent Zorbax SB C18 (150mm × 4.6mm, 5 μm)；
Flowing phase: methanol-0.1% formic acid gradient eluting, flow rate detection wavelength, with document indistinction in the same year, column temperature 35 DEG C, runs the time
60 minutes.
There were the detection method that 5 documents report is similar, World Science technology-Chinese medicine in 2015, modernize 17 (3):
HPLC method in 609-613 measures echinacoside in Cistanchis glycosides capsule, verbascoside, the content of different verbascoside simultaneously
(Yang Su), Waters C18 chromatographic column (4.6mm × 150mm, 5 μm), flow phase: methanol-0.1% formic acid solution gradient is washed
De-；Flow velocity still uses 1.0mL/min；Detection wavelength adopts the 330nm of document report in 2013；Column temperature 30 DEG C runs the time 50
Minute.In Agriculture of Anhui science, the HPLC of 43 (33): 171-173 reports measures echinacoside and verbascose in Herba Cistanches micropowder
The content (Tian Yubiao) of glycosides, Waters C18 post (4.6mm × 250mm, 5 μm), with methanol-0.1% aqueous formic acid for flowing
Phase gradient eluting；Flow velocity, detection involves column temperature and is not any change, and the operation time is 40 minutes；Chinese pharmacists 8, in 1404-1406
The one of report surveys and comments 2 kinds of active constituent contents (appointing disabled soldier) in method mensuration Herba Cistanches more, Ultimate-C18 (250mm ×
4.6mm, 5 μm)；Flowing phase: methanol (A)-0.1% formic acid (B), gradient elution；Flow velocity, detection wavelength and column temperature are the most unchanged；
45 minutes operation time.
The same year, pharmacopeia was updated to octadecylsilane chemically bonded silica as filler；With methanol as mobile phase A, with 0.1%
Formic acid solution is Mobile phase B, gradient elution；Detection wavelength is 330nm, runs 27 minutes time.
Summary of the invention
It is an object of the invention to, it is provided that a kind of quickly analysis measures echinacoside and verbascoside activity in Herba Cistanches
The method of composition.The method uses Ultra Performance Liquid Chromatography-mass spectrometric hyphenated technique to echinacoside, verbascoside in Herba Cistanches
Two kinds of active component quantitative determine, and test through methodological study, confirm this method, analyze speed and faster (complete in 5 minutes
Test), highly sensitive, favorable reproducibility, is to measure the more rapid effective means of two kinds of active component contents in Herba Cistanches simultaneously.
Echinacoside and the method for verbascoside active component in a kind of quick mensuration Herba Cistanches of the present invention, profit
Using High Performance Liquid Chromatography/Mass Spectrometry condition, concrete operations follow these steps to carry out:
A, Ultra Performance Liquid Chromatography condition: chromatographic column: Hypersil GOLD, C18 100 × 2.1mm, 1.9 μm；Flowing phase
Percent by volume is A-0.1% aqueous formic acid, B-methanol；Temporally 0min, 3.0min, 4.0min, 4.1min, 5.0min,
It is 80% by concentration respectively, 28%, 28%, 80%, the A-0.1% aqueous formic acid of 80% and concentration are 20%, 72%,
72%, 20%, the methanol solution of 20% carries out gradient elution, flow velocity: 0.2mL/min；Column temperature: room temperature；Sample size: 5 μ L；
B, Mass Spectrometry Conditions: ion source voltage :-3000v；Ion source temperature: 200 DEG C；Sheath gas: 35arb；Auxiliary gas: 8arb；
Ion transfer capillary temperature: 350 DEG C；Type ion source: electric spray ion source, verbascoside retention time 3.7 minutes, female
Ion m/z 623.154, fragments characteristic ion 162.229 and 461.374 (for quota ion)；Echinacoside retention time is
3.3 minutes, parent ion m/z 785.189, fragments characteristic ion was m/z 477.305 and 623.471 (for quota ion)；
C, the preparation of reference substance solution: precision weighs each component 1.00mg, dissolves with methanol respectively and is settled to 1mL, as
Reference substance solution storing solution；
D, the preparation of sample solution: with reference to Chinese Pharmacopoeia 2015 editions；
E, the drafting of standard curve: precision pipettes echinacoside and two kinds of active component of verbascoside, with diluent is
The water of volume ratio 80:20: methanol dilutes 10 respectively3,5×103,104,5×104,105,5×105Times, obtain mixed mark solution；Press
Chromatographic condition carries out super Ultra Performance Liquid Chromatography-mass spectral analysis；With concentration, peak area is carried out regression analysis, obtain each Component Standard
Curve, calculates regression equation；
F, precision test: continuous sample introduction 6 pin, record peak area RSD 0.59%-0.79%, relative retention time
RSD0.11%-0.13%；
G, replica test: take Herba Cistanches powder 1g, totally 7 parts, accurately weighed, extract by step d and analyze；
H, stability test: take in step d with a need testing solution, respectively at 0,2,4,6,8h sample introduction, measure wherein
Echinacoside and the content of verbascoside, and calculate RSD value；
I, sample size measure: accurate pipette samples liquid to be measured, carry out Ultra Performance Liquid Chromatography-mass spectrum by chromatographic condition and divide
Analysis, and bring peak area into standard curve, calculate echinacoside and the content of verbascoside respectively.
Accompanying drawing explanation
Fig. 1 is the spectrogram of sample in standard specimen of the present invention, and wherein RT:3.70 is Herba Cistanches effective ingredient verbascoside, RT:
3.26 is Herba Cistanches effective ingredient echinacoside；
Fig. 2 is the spectrogram in present example sample, and wherein RT:3.70 is Herba Cistanches effective ingredient verbascoside, RT:
3.27 is Herba Cistanches effective ingredient echinacoside.
Detailed description of the invention
Embodiment
Echinacoside and the method for verbascoside active component in a kind of quick mensuration Herba Cistanches of the present invention, tool
Gymnastics is made to follow these steps to carry out:
Instrument condition:
Ultra Performance Liquid Chromatography condition: chromatographic column: Hypersil GOLD, C18 100 × 2.1mm, 1.9 μm；Flowing phase:
A-0.1% aqueous formic acid；B-methanol；Elution program is as follows: 0-3 minute, A 80%-28%；3-4 minute, A 28%；4.0-
4.1 minutes A 28%-80%；4.1-5.0 minute A 80%；Flow velocity: 0.2mL/min；Column temperature: room temperature；Sample size: 5 μ L；
Mass Spectrometry Conditions: ion source voltage :-3000v；Ion source temperature: 200 DEG C；Sheath gas: 35arb；Auxiliary gas: 8arb；From
Sub-transfer capillary temperature: 350 DEG C；Electric spray ion source
The preparation of reference substance solution: precision weighs each component 1.00mg, dissolves with methanol respectively and is settled to 1mL, as right
According to product solution storing solution；
The preparation of sample solution: with reference to Chinese Pharmacopoeia 2015 editions；
The drafting of standard curve: precision pipettes echinacoside and two kinds of active component of verbascoside, is body with diluent
The long-pending water than 80:20: methanol dilutes 10 respectively3,5×103,104,5×104,105,5×105Times, obtain mixed mark solution；By color
Spectral condition carries out super Ultra Performance Liquid Chromatography-mass spectral analysis；With concentration, peak area is carried out regression analysis, obtain each Component Standard bent
Line, calculates regression equation；
Precision test: continuous sample introduction 6 pin, records peak area RSD 0.59%-0.79%, relative retention time
RSD0.11%-0.13%；Recording the response rate is 97.64%-107.36%, relative standard deviation RSD 1.56%-3.88%；
Replica test: take Herba Cistanches powder about 1g, totally 7 parts, accurately weighed, by the preparation of sample solution (with reference to China
Pharmacopeia 2015 editions) to extract and analyze, result is:
Stability test: take in replica test with a need testing solution, respectively at 0,2,4,6,8h sample introduction, measure it
Middle echinacoside and the content of verbascoside, and calculate RSD value respectively 0.6%, 0.5%；
Sample size measures: accurate pipette samples liquid to be measured, carries out Ultra Performance Liquid Chromatography-mass spectrum by chromatographic condition and divides
Analysis, and bring peak area into standard curve, the content calculating echinacoside respectively is 15.08-18.96mg/g, verbascoside
Content be 5.06-5.48mg/g.
Claims (1)
1. echinacoside and the method for verbascoside active component in a quick mensuration Herba Cistanches, it is characterised in that utilize height
Effect liquid phase chromatogram-Mass Spectrometry Conditions, concrete operations follow these steps to carry out:
A, Ultra Performance Liquid Chromatography condition: chromatographic column: Hypersil GOLD, C18 100 × 2.1mm, 1.9 μm；Flowing phase body
Long-pending percentage ratio is A-0.1% aqueous formic acid, B-methanol；Temporally 0min, 3.0min, 4.0min, 4.1min, 5.0min,
It is 80% by concentration respectively, 28%, 28%, 80%, the A-0.1% aqueous formic acid of 80% and concentration are 20%, 72%, 72%, 20%, 20%
Methanol solution carry out gradient elution, flow velocity: 0.2mL/min；Column temperature: room temperature；Sample size: 5 μ L；
B, Mass Spectrometry Conditions: ion source voltage :-3000v；Ion source temperature: 200 DEG C；Sheath gas: 35arb；Auxiliary gas: 8arb；Ion
Transfer capillary temperature: 350 DEG C；Type ion source: electric spray ion source, verbascoside retention time 3.7 minutes, parent ion
M/z 623.154, fragments characteristic ion 162.229 and 461.374；Echinacoside retention time is 3.3 minutes, parent ion m/z
785.189, fragments characteristic ion is m/z 477.305 and 623.471；
C, the preparation of reference substance solution: precision weighs each component 1.00mg, dissolves with methanol respectively and is settled to 1mL, as comparison
Product solution storing solution；
D, the preparation of sample solution: with reference to Chinese Pharmacopoeia 2015 editions；
E, the drafting of standard curve: precision pipettes echinacoside and two kinds of active component of verbascoside, is volume with diluent
Water than 80:20: methanol dilutes 10 respectively3,5×103,104,5×104,105,5×105Times, obtain mixed mark solution；By chromatograph
Condition carries out super Ultra Performance Liquid Chromatography-mass spectral analysis；With concentration, peak area is carried out regression analysis, obtain each Component Standard bent
Line, calculates regression equation；
F, precision test: continuous sample introduction 6 pin, record peak area RSD 0.59%-0.79%, relative retention time RSD 0.11%-
0.13%；
G, replica test: take Herba Cistanches powder 1g, totally 7 parts, accurately weighed, extract by step d and analyze；
H, stability test: take in step d with a need testing solution, respectively at 0,2,4,6,8h sample introduction, measure wherein strobile
Echinacoside and the content of verbascoside, and calculate RSD value；
I, sample size measure: accurate pipette samples liquid to be measured, carry out Ultra Performance Liquid Chromatography-mass spectral analysis by chromatographic condition,
And bring peak area into standard curve, calculate echinacoside and the content of verbascoside respectively.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109507319A (en) * | 2018-11-20 | 2019-03-22 | 中国科学院新疆生态与地理研究所 | A kind of method of resveratrol activity component content in quick measurement tree peony fruit pod |
| CN109991328A (en) * | 2019-04-04 | 2019-07-09 | 西安医学院 | A kind of affine cudweed one surveys the quality evaluating method more commented |
| CN110092805A (en) * | 2019-06-12 | 2019-08-06 | 劲牌有限公司 | The extracting method of echinacoside and acteoside in Herba Cistanches |
| CN112630327A (en) * | 2020-12-03 | 2021-04-09 | 武汉职业技术学院 | Quick detection method for active ingredients of cistanche |
| CN115128200A (en) * | 2022-07-26 | 2022-09-30 | 吉首大学 | HPLC (high Performance liquid chromatography) quality detection method for paulownia leaves |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109507319A (en) * | 2018-11-20 | 2019-03-22 | 中国科学院新疆生态与地理研究所 | A kind of method of resveratrol activity component content in quick measurement tree peony fruit pod |
| CN109991328A (en) * | 2019-04-04 | 2019-07-09 | 西安医学院 | A kind of affine cudweed one surveys the quality evaluating method more commented |
| CN109991328B (en) * | 2019-04-04 | 2022-04-22 | 西安医学院 | Quality evaluation method for one-test-multiple evaluation of affine cudweed |
| CN110092805A (en) * | 2019-06-12 | 2019-08-06 | 劲牌有限公司 | The extracting method of echinacoside and acteoside in Herba Cistanches |
| CN110092805B (en) * | 2019-06-12 | 2020-11-13 | 劲牌有限公司 | Method for extracting echinacoside and verbascoside from cistanche |
| CN112630327A (en) * | 2020-12-03 | 2021-04-09 | 武汉职业技术学院 | Quick detection method for active ingredients of cistanche |
| CN115128200A (en) * | 2022-07-26 | 2022-09-30 | 吉首大学 | HPLC (high Performance liquid chromatography) quality detection method for paulownia leaves |
| CN115128200B (en) * | 2022-07-26 | 2024-01-19 | 吉首大学 | HPLC quality detection method for paulownia tomentosa leaves |
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