CN110297061A - Survey the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content using one more - Google Patents
Survey the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content using one more Download PDFInfo
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Abstract
The present invention relates to traditional Chinese medicine ingredients detection technique fields, more particularly to a kind of the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content are surveyed using one more, include the following steps: that preparation (3) chromatographic condition of preparation (2) test solution of (1) reference substance solution and system suitability (4) measure.The present invention uses chlorogenic acid to carry out a survey for internal reference object and comments more, establishes the relative correction factor between chlorogenic acid and caffeic acid and galuteolin, passes through relative correction factor and calculate Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content.The method of the present invention is simple and convenient to operate, practicability is high, as a result accurate, the Simultaneous Determination of Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin component content is able to achieve using this method, to the quality of effective evaluation and control Chinese ixeris herb medicinal material, to guarantee that clinical application safety and effective and its herb resource exploitation provide scientific basis.
Description
Technical field
The present invention relates to traditional Chinese medicine ingredients detection technique fields, and in particular to a kind of to survey comment in method measurement Chinese ixeris herb using one more
The method of chlorogenic acid, caffeic acid and galuteolin content.
Background technique
Chinese ixeris herb (Ixeridiumchinense(Thunb.) Tzvel.) it is the small gutweed category herbaceos perennial of composite family,
All herbal medicine, alias China denticulate ixeris herb, tall lattuce flower and leaf, small Chinese Ixeris etc. are distributed in northern, south and east provinces and regions, it is not only pharmaceutically acceptable but also
It is edible, have clearing heat and detoxicating, anti-inflammatory cool blood, alleviating pain and detumescence, antitumor and other effects, for treat nameless gall, peritoneal abscess,
The diseases such as dysentery, ecphyaditis, pneumonia, arthritis.
Liu Chang etc. go deep into the chemical component of tall lattuce flower and leaf containing flavones ingredient in systematic research discovery tall lattuce flower and leaf,
Wherein flavones ingredient has very strong pharmacological activity；It mainly contains: luteolin, luteolin -7-O- β-D-Glucose glycosides,
Api-genin-7-O-β-D-glucoside, rye grass element, luteolin -7-O- β-D-Glucose glycosides acetic acid esters, apiolin also contain
Having caffeic acid etc., (Liu Chang, Yan Mingming, Shao Shuai wait the Chinese experimental side progress of tall lattuce flower and leaf chemical component and pharmacological action
Agent magazine, 2015).All peaches etc. establish galuteolin content in measurement Chinese ixeris herb to preferably utilize Chinese ixeris herb resource
HPLC method, and determine the content of galuteolin in 6 different sources Chinese ixeris herbs, obtain wooden in 6 different sources Chinese ixeris herbs
The content of rhinoceros grass glycosides be 1.2812.288 mgg-1, average content be 1.780 mgg-1(weeks peaches, Sui Lin, Na Shengsang,
Content [J] pharmacy today of galuteolin in equal .HPLC method measurement different sources Chinese ixeris herb, 2018).Zhou Honglei etc. is to China
The chemical component of denticulate ixeris herb alias (Chinese ixeris herb) is studied, isolated 5 kinds of compounds (Zhou Honglei, Yuan Jiu from alcohol extract
Research [J] Chinese herbal medicine of flourish China denticulate ixeris herb chemical component, 1996).
In terms of pharmacological research, Tai Wenjie etc. show that Chinese ixeris herb has a variety of pharmacological activity, including anti-inflammatory liver protection acts on, and resists
Virus, leukemia resisting action, treatment diabetes effect etc.；From chemical constitution study, containing there are many flavones in Chinese ixeris herb
Class compound, has antitumor, anti-oxidant etc., and under optimal extract process, extracts and obtains Chinese ixeris herb general flavone, and adopt
With the content of visible spectrophotometry measurement Chinese ixeris herb general flavone, the results showed that in rutin concentration range in it is good it is linear (too
The extraction of general flavone and the Guizhou antioxidant activity research [J] college of education journal in Wen Jie, Chen Shengke, Gan Xiuhai Chinese ixeris herb,
2016).Chinese denticulate ixeris herb alias (Chinese ixeris herb) the extract C hinensiolide A of the prime reports such as Zhou multitude in vitro can be effective
Inhibition Lung Adenocarcinoma A 549 Cell, liver cancer Ble-7402 cell and LoVo cell growth, have stronger anti-tumor activity
(Zhou Li, Zhao Ying, Wang Yu wait traditional Chinese medical science in anti tumor activity in vitro [J] of China Herba Ixeritis Denticulatae extract Chinensiolide A
It learns and innovates, 2015).Wang Lulu etc. has found that Chinese denticulate ixeris herb alias (Chinese ixeris herb) has good antiviral and drop under study for action
The effects of low blood lipid and cholesterol, (Wang Lulu, Yang Li, Li Yuehua waited China denticulate ixeris herb general flavone and total triterpene extraction process excellent
Change and studies the Guangdong [J] chemical industry, 2011).Magnificent denticulate ixeris herb alias (Chinese ixeris herb) middle discovery discovery China is bitter under study for action for Cheng Liang China etc.
Mai dish extract has significant hypoglycemic effect；Discovery contains chlorogenic acid ingredient during this experiment, and to the ingredient
It is studied and is explored；Illustrate that Chinese ixeris herb contains a large amount of flavones ingredient, and plays the role of very big pharmacological treatment (Cheng Liang
The preparation of magnificent China denticulate ixeris herb extractive of general flavone and the drug effect and the Heilungkiang Mechanism Study [D] Chinese medicine for treating diabetes B
Medicine university, 2016).
It is above-mentioned as it can be seen that Chinese ixeris herb be it is important in China's Chinese medicine and it is common simply, medical value is very high, and its quality is good
It is bad closely related with people's health interests.It is many to influence Chinese medicine qualitative factor, causes Chinese medicine quality control in the market tired
Difficulty, it is only excessively unilateral with single component evaluation Chinese medicine quality at present, it is superior in quality Chinese medicine cannot to be reacted very well.Due to plant
The complexity of chemical component, multi objective assay have become the common recognition of plant origin Control of drug quality.With tradition measurement
Method measures several ingredients in Chinese medicine simultaneously, not only faces that reference substance is rare, and round of visits is long, also energy consumption, time-consuming.One survey is more
Comment method by existing intrinsic function relationship and proportionate relationship method between effective component of chinese medicine, measuring an ingredient, (reference substance is easy
, cheap, responder) realize multiple ingredients (reference substance be difficult to obtain or difficult supplier) Simultaneous Determination；In not only solving
Medicine multicomponent is quantitatively and reference substance present in the control of multi objective quality lacks problem, while realizing that multi objective synchronizing quality controls
React traditional Chinese medicine quality.Correlative study at present about the quality analysis of Chinese ixeris herb is less, through consulting literatures, currently there is no both at home and abroad
One surveys the reports for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content more.
Summary of the invention
The purpose of the present invention is provide a kind of survey using one in view of the deficiencies of the prior art to comment method to measure Chinese ixeris herb more
(Ixeridiumchinense(Thunb.) Tzvel.) Content of Chlorogenic Acid, caffeic acid and galuteolin content method, this method
Simply, accurately, easy to operate, practicability is high, as a result accurately, using this method be able to achieve Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and
The Simultaneous Determination of galuteolin component content, thus effective evaluation and control Chinese ixeris herb (Ixeridiumchinense(Thunb.)
Tzvel.) the quality of medicinal material, to guarantee that clinical application safety and effective and its herb resource exploitation provide scientific basis.
To achieve the above object, the present invention adopts the following technical scheme:
It surveys the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content using one more, includes the following steps:
(1) preparation of reference substance solution: precision weighing chlorogenic acid reference substance 11.59mg, caffeic acid reference substance 6.72mg, reseda
Glycosides reference substance 13.60mg, is separately added into 25mL volumetric flask, and proper amount of methanol is added to be diluted to scale, and ultrasound makes it dissolve, and shakes up,
Precision measures above-mentioned solution of chlorogenic acid 1.5mL, coffee acid solution 1.5mL and galuteolin solution 6.0mL, sets same 10mL capacity
It in bottle and with methanol constant volume to scale, shakes up to get mixed reference substance solution, containing green in the every 1mL solution of mixed reference substance solution
66.9 μ g of ortho acid, 40.35 μ g of caffeic acid, 306.78 μ g of galuteolin；
(2) preparation of test solution: taking Chinese ixeris herb medicinal powder 2g, be placed in 100mL conical flask, and volumetric concentration is added and is
70% methanol 30mL, weighed weight, ultrasonic extraction 60min, 30 DEG C of temperature, frequency 70kHz is let cool, and is 70% with volumetric concentration
Methanol supply the weight of less loss, filtering, take subsequent filtrate cross 0.45 μm of miillpore filter to get；
(3) chromatographic condition and system suitability: chromatographic column: II SIZE-C of CAPCELLPAK TYPE MG18(4.6 ×
250mm, 5 μm)；Volume flow is 1.0mlmin-1；Detection wavelength is 330nm；Column temperature is 25 DEG C；Sample volume is 5 μ L；Flowing
Gradient elution is mutually carried out using -0.2% phosphate aqueous solution of methanol, elution program is shown in Table 1, and theoretical cam curve is calculated by chlorogenic acid peak
5000 should be not less than；
(4) it measures: it is accurate respectively to draw reference substance solution and each 5 μ L of test solution, liquid chromatograph is injected, in above system
It measures under the chromatographic condition of adaptability, is calculated using internal standard method, test solution Content of Chlorogenic Acid, caffeic acid and galuteolin chromatography
Peak is consistent with chromatographic peak retention time of reference substance, and is greater than 1.5 with other separating degrees that ingredient coexists, and main peak reaches with impurity peaks
Baseline separation；The peak value that chlorogenic acid, caffeic acid and galuteolin are presented in the characteristic spectrum of test solution, is interior with chlorogenic acid
Join object, calculate the relative correction factor of chlorogenic acid and caffeic acid and galuteolin, is calculated in Chinese ixeris herb by relative correction factor
Chlorogenic acid, caffeic acid and galuteolin content.
The methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content, institute are surveyed in above-mentioned use one more
The calculation method for the relative correction factor stated are as follows: aspiration step (1) is prepared respectively mixed reference substance solution 0.4,0.6,
0.8,1.0,1.2,1.6 mL is set respectively in 2.0 mL measuring bottles, is added the methanol constant volume that volumetric concentration is 70% to scale, is shaken up, make
It at the serial mixed reference substance solution of various concentration, is measured by chromatographic condition, respectively 5 μ L of sample introduction, using chlorogenic acid as internal standard, measurement
The peak area of caffeic acid, galuteolin in Chinese ixeris herb, according to relative correction factor calculation formula fac=f a/fc=(Aa/Ca)/
(Ac/Cc) calculates the relative correction factor fac of normal caffeic acid, galuteolin, is calculated in formula with reference substance, and Aa is internal reference object
The peak area of chlorogenic acid, Ca are the concentration of internal reference object chlorogenic acid, and Ac is certain Component peak area to be measured;Cc is that certain ingredient to be measured is dense
Degree；Relative correction factor fac is the average value of each experiment the data obtained.
The invention has the benefit that
1, the present invention surveys the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content using one more, meets
The requirement of methodology validation, in the case where reference substance shortage, method of the invention can be applied to the quality evaluation of Chinese ixeris herb,
The content of Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin is measured simultaneously, is not only simple, fast, and reproducible, it can be with
The loss to economize on resources reduces solvent consumption and analysis time, environmental protection is more advantageous to, for the quality for establishing white melitot medicinal material
Evaluation provides foundation.
2, a kind of survey using one provided by the invention comments method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin to contain more
The method of amount, this method is simple, measurement is accurate, and easy to operate, practicability is high, and as a result accurately, method repeatability, durability is good,
It is able to achieve the Simultaneous Determination of Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin component content, using this method so as to more
Good, more comprehensively effectively evaluating and control Chinese ixeris herb medicinal material quality, can be used in the quality control to Chinese ixeris herb medicinal material, to protect
It demonstrate,proves clinical application safety and effective and its herb resource exploitation provides scientific basis；Evade simultaneously by mostly at assay band
The cost and operational issue come.
Detailed description of the invention
Fig. 1 is chlorogenic acid canonical plotting；
Fig. 2 is caffeic acid canonical plotting；
Fig. 3 is galuteolin canonical plotting；
Fig. 4 is to mix reference substance chromatogram, in figure, 1, chlorogenic acid, and 2, caffeic acid, 3, galuteolin；
Fig. 5 is Chinese ixeris herb sample chromatogram figure, 1, chlorogenic acid, 2, caffeic acid, 3, galuteolin.
Specific embodiment
Embodiment 1
Survey the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content to 10 batches of mountain hardships using above-mentioned one more
Mai medicinal material is detected, and specific measurement situation is as follows:
1 experimental material, instrument and reagent
1.1 experimental material
Chinese ixeris herb Chinese medicine harvesting area and date are shown in Table 2, are accredited as the small hardship of composite family through Guangxi University of Chinese Medicine professor Wei Songji
Mai platymiscium Chinese ixeris herb (Ixeridiumchinense(Thunb.) Tzvel.) herb.
1.2 instrument
U3000 high performance liquid chromatograph (Thermo Fisher company, the U.S.)；1260 high performance liquid chromatography discuss the (U.S.
Agilent company)；II SIZE C18 of chromatographic column CAPCELLPAK TYPE (4.6mmI.D. × 250mm, 5 μm), Agilent
Eclipse plus C18 (4.6 × 250mm, 5 μm), Waters Atlantis T3 (4.6 × 250mm, 5 μm)；XS-205Du
Electronic analytical balance (METTLER TOLEDO company, Switzerland)；(city of Kunshan's ultrasonic instrument is limited for KQ-500GDV ultrasonic cleaner
Company)；501 thermostatic water-circulator bath pots (Medical Instruments factory, Jintan City)；Synergy ultrapure water machine (Millipore company, the U.S.).
1.3 reagent
Chlorogenic acid (110753-201415, content is in terms of 96.2%), caffeic acid (110885-200102), luteolin
(111720-201307 content is in terms of 94.0%) is purchased from National Institute for Food and Drugs Control.Acetonitrile, methanol (chromatographically pure, beauty
Fisher company, state), phosphoric acid (lot number: 20150327, fine chemistry industry research institute, excellent pure grade are recovered in Tianjin), effect liquid phase chromatogram
View is ultrapure water with water, other agents useful for same are that analysis is pure.
2 experimental methods and result
2.1 survey the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content using one more, including as follows
Step:
(1) preparation of reference substance solution: precision weighing chlorogenic acid reference substance 11.59mg, caffeic acid reference substance 6.72mg, reseda
Glycosides reference substance 13.60mg, is separately added into 25mL volumetric flask, and proper amount of methanol is added to be diluted to scale, and ultrasound makes it dissolve, and shakes up,
Precision measures above-mentioned solution of chlorogenic acid 1.5mL, coffee acid solution 1.5mL and galuteolin solution 6.0mL, sets same 10mL capacity
It in bottle and with methanol constant volume to scale, shakes up to get mixed reference substance solution, containing green in the every 1mL solution of mixed reference substance solution
66.9 μ g of ortho acid, 40.35 μ g of caffeic acid, 306.78 μ g of galuteolin；
(2) preparation of test solution: taking Chinese ixeris herb medicinal powder 2g, be placed in 100mL conical flask, and volumetric concentration is added and is
70% methanol 30mL, weighed weight, ultrasonic extraction 60min, 30 DEG C of temperature, frequency 70kHz is let cool, and is 70% with volumetric concentration
Methanol supply the weight of less loss, filtering, take subsequent filtrate cross 0.45 μm of miillpore filter to get；
(3) chromatographic condition and system suitability: chromatographic column: II SIZE-C of CAPCELLPAK TYPE MG18(4.6 ×
250mm, 5 μm)；Volume flow is 1.0mlmin-1；Detection wavelength is 330nm；Column temperature is 25 DEG C；Sample volume is 5 μ L；Flowing
Gradient elution is mutually carried out using -0.2% phosphate aqueous solution of methanol, elution program is shown in Table 1, and theoretical cam curve is calculated by chlorogenic acid peak
5000 should be not less than；
(4) it measures: it is accurate respectively to draw reference substance solution and each 5 μ L of test solution, liquid chromatograph is injected, in above system
It measures under the chromatographic condition of adaptability, is calculated using internal standard method, test solution Content of Chlorogenic Acid, caffeic acid and galuteolin chromatography
Peak is consistent with chromatographic peak retention time of reference substance, and is greater than 1.5 with other separating degrees that ingredient coexists, and main peak reaches with impurity peaks
Baseline separation；The peak value that chlorogenic acid, caffeic acid and galuteolin are presented in the characteristic spectrum of test solution, is interior with chlorogenic acid
Join object, calculate the relative correction factor of chlorogenic acid and caffeic acid and galuteolin, is calculated in Chinese ixeris herb by relative correction factor
Chlorogenic acid, caffeic acid and galuteolin content.
The methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content, institute are surveyed in above-mentioned use one more
The calculation method for the relative correction factor stated are as follows: aspiration step (1) is prepared respectively mixed reference substance solution 0.4,0.6,
0.8,1.0,1.2,1.6 mL is set respectively in 2.0 mL measuring bottles, is added the methanol constant volume that volumetric concentration is 70% to scale, is shaken up, make
It at the serial mixed reference substance solution of various concentration, is measured by chromatographic condition, respectively 5 μ L of sample introduction, using chlorogenic acid as internal standard, measurement
The peak area of caffeic acid, galuteolin in Chinese ixeris herb, according to relative correction factor calculation formula fac=f a/fc=(Aa/Ca)/
(Ac/Cc) calculates the relative correction factor fac of normal caffeic acid, galuteolin, is calculated in formula with reference substance, and Aa is internal reference object
The peak area of chlorogenic acid, Ca are the concentration of internal reference object chlorogenic acid, and Ac is certain Component peak area to be measured;Cc is that certain ingredient to be measured is dense
Degree；Relative correction factor fac is the average value of each experiment the data obtained.
2.2 present invention one survey the side of the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content more
The science of law is investigated as follows:
2.2.1 the investigation result of the range of linearity
0.4,0.6,0.8,1.0,1.2,1.6 mL of mixed reference substance solution that step (1) of the present invention is prepared is drawn respectively,
It is set in 2.0 mL measuring bottles respectively, 70% methanol constant volume is added to shake up, the serial mixed reference substance solution of various concentration is made to scale,
It is measured by chromatographic condition defined by the present invention, respectively 5 μ L of sample introduction, using peak area as ordinate, concentration is that abscissa is linear
It returns range investigation and is shown in Table 3, the result is shown in Figure 1,2,3.Can be obtained by result: the range of linearity is investigated good in this experiment.
2.2.2 precision test
2 g of S4 Chinese ixeris herb powder is taken, prepares Chinese ixeris herb test solution by the preparation method of test solution of the present invention, according to
It chromatographic condition continuous sample introduction six times, records sample map and calculates Chinese ixeris herb peak area.The results are shown in Table 4: chlorogenic acid, caffeic acid and
Galuteolin average peak area value is 15.617,5.467 and 31.040, and RSD is respectively 2.0%, 2.1% and 2.1%, knot
The precision that fruit can obtain instrument is good.
2.2.3 repetitive test
6 parts of S4 Chinese ixeris herb powder is taken, every part of 2 g prepare test sample by the preparation method of test solution of the present invention, according to color
Spectral condition is measured, and observation map peak and record Chinese ixeris herb sample data calculate Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid, wood
The content of rhinoceros grass glycosides.The results are shown in Table 5: chlorogenic acid average content is 1.167mg/g, and RSD is 1.7 %, and caffeic acid average content is
0.222mg/g, RSD 2.3%, galuteolin average content are 2.510mg/g, RSD 2.3%.Show the repeatability of this method
Well.
2.2.4 stability test
A test solution in repetitive test is taken, is measured for 24 hours according to chromatographic condition 1,2,4,6,8,12, mountain is recorded
Gutweed sample map and peak area simultaneously calculate Chinese ixeris herb and measure content data, investigate the stability of test solution.It the results are shown in Table
6: chlorogenic acid average content 1.146mg/g, RSD are 5.1 %, and caffeic acid average content is 0.213mg/g, RSD 4.8%, sweet-scented osmanthus
Careless glycosides average content is 2.416mg/g, RSD 5.1%.It obtains and has good stability in test solution interior for 24 hours.
2.2.5 it is loaded recovery test
The accurately weighed content (1.167mg containing chlorogenic acid, caffeic acid 0.222mg, galuteolin 2.510mg in every 1g) of having measured
6 parts of Chinese ixeris herb sample, every part of 1g；Another precision weighs chlorogenic acid reference substance 6.86mg, caffeic acid reference substance 1.34mg, galuteolin
Reference substance 16.24mg is set in 200ml measuring bottle, adds appropriate 70% methanol ultrasound to make to dissolve, and be diluted to scale, is shaken up to get mixed
Close reference substance stock solution (containing 33.0 μ g of chlorogenic acid, 6.7 μ g of caffeic acid, 76.3 μ g of galuteolin in every 1ml).It is accurate respectively to be added
Reference substance stock solution 30ml is measured, and remember by Chinese ixeris herb sample-adding recovery sample solution is prepared under " 2.5 " item according to chromatographic condition
Record chlorogenic acid, caffeic acid, galuteolin peak area and calculate the rate of recovery, RSD < 3 can be obtained by result.It the results are shown in Table 7,8,9.
The calculating of index relative correction factor to be measured in 2.3 Chinese ixeris herbs
Under the chromatographic condition that the present invention limits, precision draws the mixing control under " the investigation result of the 2.2.1 range of linearity " item
Product solution measures the peak area of caffeic acid, galuteolin in Chinese ixeris herb, according to relative correction factor meter using chlorogenic acid as internal standard
The relative correction factor that formula calculates normal caffeic acid, galuteolin is calculated, the results are shown in Table 10, relative correction factor is averaged.It presses
Following formula calculates, and: fac=fa/fc=(Aa/Ca)/(Ac/Cc) is calculated with reference substance in formula, Aa and be internal reference object (chlorogenic acid)
Peak area, Ca are internal reference object (chlorogenic acid) concentration, and Ac is certain Component peak area to be measured；Cc is certain constituent concentration to be measured.
The investigation of 2.4 system robustness
It is measured under the chromatographic condition that the present invention limits, precision draws mixing pair under " the investigation result of the 2.2.1 range of linearity " item
According to product solution, relative correction factor, the relative retention value of component content to be measured in reference substance are calculated to investigate durability, and investigate
Chromatographic positioning can preferably be evaluated and tested obtained as a result, commenting method in application from now on so that one survey of verifying.
2.4.1 different sample volumes are investigated
Under " 2.4 " item, the influence of 4,5,6 μ L difference sample volumes is investigated, the results are shown in Table 11.Different volumes flow influences not it
Significantly, good tolerance.
2.4.2 different instruments, chromatographic column are investigated
Under the conditions of " 2.4 " item, ThermoUltiMate 3000, II SIZE of Agilrnt 1260 and YPE MG-are selected
C18, Agilent Eclipse plus- C18, Atlantis T3-C18 chromatographic column (5 μm of 4.6 × 250mm) are investigated opposite
The influence of correction factor, the results are shown in Table 12.Know that different instruments, chromatographic column are unobvious to its influence degree, good tolerance.
2.4.3 different volumes flow is investigated
Under " 2.4 " item, investigate 0.9,1.0,1.1ml/min volume flow is to the shadow of relative correction factor and relative retention value
It rings, the results are shown in Table 13.Different volumes flow is unobvious to its influence degree, good tolerance.
2.4.4 different column temperatures are investigated
Under " 2.4 " item, 20,25,30, influence of DEG C column temperature to relative correction factor and relative retention value are investigated, the results are shown in Table
14.Different column temperatures are unobvious to its influence degree, good tolerance.
2.4.5 different wave length is investigated
Under " 2.4 " item, influence of 220,254, the 330 nm Detection wavelengths to relative correction factor and relative retention value, knot are investigated
Fruit is shown in Table 15.Its Detection wavelength is unobvious to its influence degree, good tolerance.
2.5 1 survey the positioning for commenting method chromatographic peak to be measured more
Under " 2.4 " item, the influence of the relative retention value under " 2.4.2 " item in different instruments, chromatographic column is investigated, the results are shown in Table
16.As seen from table, relative retention value fluctuation is smaller, and the RSD of caffeic acid and galuteolin is respectively 1.4% and 6.8%, therefore is selected
Different instruments, chromatographic column relative retention value are as chromatographic peak positioning index.
2.6 1 surveys comments method compared with external standard method assay result
After methodology optimizes and investigates, is surveyed using established above one and comment method more, to the coffee in 10 batches of Chinese ixeris herb extracts
Coffee acid, galuteolin ingredient content be determined, by reference substance solution under " 2.2.1 " item, measurement side according to the invention
The measurement of method sample introduction, measures the content (Fig. 4, Fig. 5) of caffeic acid, galuteolin respectively.Using external standard method and relative correction factor into
Row compares, and the results are shown in Table 17.
The accuracys for commenting method " QAMS " method are surveyed for verifying one, to the measurement result and external standard method knot of " QAMS " method more
Fruit compares.Survey the feasibilities for commenting method with accuracy (Accuracy) to evaluate established one more, accuracy by with
Lower formula calculates: accuracy (%)=CS/CE × 100%, wherein and CS indicates that a survey comments method to calculate resulting content more,
CE indicates to calculate resulting content by external standard method.
Experimental result: this experiment using one survey comment method measure 10 batches of place of production Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and
The content of galuteolin with the higher place of production of chlorogenic acid content is being as the result is shown S10, S6, S4, the higher production of coffee acid content
Ground be S10, S5, S6, S4, S2, the higher place of production of galuteolin content be S10, S5, S4, S6, therefrom it can be concluded that S10, S6,
The content of the Chinese ixeris herb chlorogenic acid in the place of production S4, caffeic acid and galuteolin is higher.
3 discuss
The selection of 3.1 mobile phases
This experiment is to investigate the content of Chinese ixeris herb extract, so that it is determined that contained each ingredient, can obtain through consulting literatures, is selected
0.1% phosphoric acid: acetonitrile；0.1% phosphoric acid: methanol；0.2% phosphoric acid: acetonitrile；0.2% phosphoric acid: methanol；Water: the various mobility such as methanol into
Row is investigated；In experimentation discovery because the polarity of water it is larger, appearance ingredient is less and stack up between peak and peak, it is contemplated that
Acid adding；Separating effect is not achieved when with 0.1% phosphoric acid, therefore selects 0.2% phosphoric acid；When selecting acetonitrile and methanol, acetonitrile is being separated
When cutting edge of a knife or a sword shape it is relatively narrow and separating effect is poor, select 0.2% phosphoric acid: separating effect is preferable when methanol uses gradient elution.
The preparation of 3.2 test samples
The preparation of test sample is determined by investigating extraction conditions, in Different Extraction Method, different solvents concentration methanol, different material
Liquor ratio and different extraction times, the solution extracted is direct to cross miillpore filter, and direct injected after filtering tests discovery with 70%
Obtained peak base is steady after methanol 30ml ultrasound 60min, and detection ingredient is more in chromatogram and peak shape is preferable.It is investigating back
It is found when stream method and ultrasonic method, less, consulting literatures discovery is more to like using ultrasonic extraction for the content difference that the two is extracted.
The selection of 3.3 Detection wavelengths
This experiment determines mobility when investigating sample, chromatographic condition, determined using reference substance in Chinese ixeris herb containing chlorogenic acid,
Caffeic acid and galuteolin, and to determining reference substance and medicinal powder with hair when determining chromatographic condition progress full wavelength scanner
Present 330nm has shared peak to occur, and retention time is identical, and chromatographic peak profile is also preferable, so using 330nm as detection wave
It is long.
The screening of 3.4 internal standards
Chinese ixeris herb is calculating 3 kinds of contents in Chinese ixeris herb with external standard method due to more containing more flavones ingredient in this experiment
Shi Faxian, the content of chlorogenic acid and the content of 3 kinds of ingredients are more stable, under conditions of limit has reference substance in measured three contents
Chlorogenic acid reference substance is easier to buy, acquired height, using chlorogenic acid as internal reference object, establishes the opposite school of its caffeic acid and galuteolin
Positive divisor.And experiment discovery, chlorogenic acid appearance time is moderate on different instruments and chromatographic column and the poor fluctuation of retention time is smaller,
RSD is within 5%；It is more unstable since content is lower in caffeic acid, Systematic Analysis should not be carried out；And the guarantor of galuteolin
It stays the time to be greater than 5%, therefore the positioning between each component should not be carried out.So selecting chlorogenic acid for internal standard, with the phase in " QAMS " method
The content of caffeic acid and galuteolin is calculated correction factor.
In conclusion the content assaying method of this experiment foundation is easy to operate, result is accurate, it can be used for green original in Chinese ixeris herb
The Simultaneous Determination of acid, caffeic acid and galuteolin component content
The investigation of 3.5 " QAMS " method durabilities
This experiment investigates Ultimate3000 and Agilent1260 using the selection of " QAMS " method, and uses different chromatographic conditions,
The durability of the method is investigated, influence of the measured each ingredient to relative correction factor and relative retention value verifies this
Durability of the method in the control of Chinese ixeris herb quality.The result shows that: it can be in the case where reference substance shortage, using " one surveys comment more
Repeatability loss that is more satisfactory, and can economizing on resources in method ".
The positioning of 3.6 chromatographic peaks
This experiment is can to survey using one when only using single reference substance and comment method to be properly positioned measured coffee in Chinese ixeris herb more
The problem of 2 kinds of acid, luteolin ingredient chromatographic peaks, selects the poor retention time between each ingredient, relative retention value, separating degree to make
For localization criteria, and the two is investigated with different instruments, chromatographic column.The result shows that relative retention value can more accurately determine
Position chromatographic peak.
3.7 external standard methods survey the evaluations for commenting method with one more
Analyzed by the content measured by 10 batches of place of production Chinese ixeris herb medicinal materials, with external standard method to one survey comment the result of method into
Row compares, and obtained result is much the same, the results showed that, one comments more survey methods simplicity, accuracy high, with higher feasible
Property.
4 conclusions
Using external standard method and one survey comment method to be compared 10 batches of Chinese ixeris herb medicinal materials, obtaining in the result, before each place of production
There were significant differences for existing content, some different local contents are higher, some contents are lower, illustrate the important of genunie medicinal materials
Property；It is simple using this method, accurate, it is easy to operate, so assay can be used in the case where reference substance shortage, while can
Solvent consumption and analysis time are reduced, environmental protection is more advantageous to.
Claims (2)
1. surveying the methods for commenting method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content using one more, which is characterized in that
Include the following steps:
(1) preparation of reference substance solution: precision weighing chlorogenic acid reference substance 11.59mg, caffeic acid reference substance 6.72mg, reseda
Glycosides reference substance 13.60mg, is separately added into 25mL volumetric flask, and proper amount of methanol is added to be diluted to scale, and ultrasound makes it dissolve, and shakes up,
Precision measures above-mentioned solution of chlorogenic acid 1.5mL, coffee acid solution 1.5mL and galuteolin solution 6.0mL, sets same 10mL capacity
It in bottle and with methanol constant volume to scale, shakes up to get mixed reference substance solution, containing green in the every 1mL solution of mixed reference substance solution
66.9 μ g of ortho acid, 40.35 μ g of caffeic acid, 306.78 μ g of galuteolin；
(2) preparation of test solution: taking Chinese ixeris herb medicinal powder 2g, be placed in 100mL conical flask, and volumetric concentration is added and is
70% methanol 30mL, weighed weight, ultrasonic extraction 60min, 30 DEG C of temperature, frequency 70kHz is let cool, and is 70% with volumetric concentration
Methanol supply the weight of less loss, filtering, take subsequent filtrate cross 0.45 μm of miillpore filter to get；
(3) chromatographic condition and system suitability: chromatographic column: II SIZE-C of CAPCELLPAK TYPE MG18；Volume flow
For 1.0mlmin-1；Detection wavelength is 330nm；Column temperature is 25 DEG C；Sample volume is 5 μ L；Mobile phase uses -0.2% phosphoric acid of methanol
Aqueous solution carries out gradient elution, and elution program is shown in Table 1, and theoretical cam curve is calculated by chlorogenic acid peak is not less than 5000；
(4) it measures: it is accurate respectively to draw reference substance solution and each 5 μ L of test solution, liquid chromatograph is injected, in above system
It measures under the chromatographic condition of adaptability, is calculated using internal standard method, chlorogenic acid, caffeic acid are presented in the characteristic spectrum of test solution
The relative correction factor of chlorogenic acid and caffeic acid and galuteolin is calculated using chlorogenic acid as internal reference object with the peak value of galuteolin,
Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin content are calculated by relative correction factor.
2. according to claim 1 survey using one comments method measurement Chinese ixeris herb Content of Chlorogenic Acid, caffeic acid and galuteolin to contain more
The method of amount, which is characterized in that the calculation method of the relative correction factor are as follows: aspiration step (1) is prepared respectively
0.4,0.6,0.8,1.0,1.2,1.6 mL of mixed reference substance solution, sets respectively in 2.0 mL measuring bottles, and adding volumetric concentration is 70%
Methanol constant volume shakes up to scale, the serial mixed reference substance solution of various concentration is made, measure by chromatographic condition, respectively sample introduction 5
μ L measures the peak area of caffeic acid, galuteolin in Chinese ixeris herb using chlorogenic acid as internal standard, calculates according to relative correction factor public
Formula fac=f a/fc=(Aa/Ca)/(Ac/Cc) calculates the relative correction factor fac of normal caffeic acid, galuteolin, uses in formula
Reference substance calculates, and Aa is the peak area of internal reference object chlorogenic acid, and Ca is the concentration of internal reference object chlorogenic acid, and Ac is certain ingredient to be measured
Peak area;Cc is certain constituent concentration to be measured；Relative correction factor fac is the average value of each experiment the data obtained.
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