CN109828040A - The construction method and detection method of eclipta medicinal material UPLC characteristic spectrum - Google Patents
The construction method and detection method of eclipta medicinal material UPLC characteristic spectrum Download PDFInfo
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Abstract
The present invention relates to a kind of eclipta medicinal material UPLC characteristic spectrum construction method and detection methods.This feature map construction method is the following steps are included: prepare reference substance reference solution using wedelolactone and bis--O- caffeoylquinic acids of 4,5- as reference substance respectively；Control medicinal material reference solution is prepared with eclipta control medicinal material；Eclipta medicinal material and standard decoction sample are taken respectively, Extraction solvent is added to extract, and are filtered, gained filtrate is as test solution and standard decoction test solution；The reference solution, test solution, standard decoction test solution are drawn respectively, inject Ultra Performance Liquid Chromatography instrument, measurement；Compare gained test sample map and standard decoction test sample map, demarcate the characteristic peak of 8 water soluble ingredients, obtains the UPLC characteristic spectrum of eclipta medicinal material.This feature map can be used for the quality of qualitative and quantitative analysis eclipta medicinal material, it can be ensured that using the quality of the eclipta traditional decoction of medicinal material preparation, be also applied for detecting other preparations containing eclipta.
Description
Technical field
The present invention relates to pharmaceutical technology fields, more particularly to a kind of construction method of eclipta medicinal material UPLC characteristic spectrum
And detection method.
Background technique
Eclipta nickname Eclipta prostrata, Nanjing grass, WUXINCAO, white flower wedelia chinensis etc..With original name Eclipta prostrata, first recorded in " Tang's sheet
Grass ", it is the dry aerial parts of compositae plant Eclipta prostrata Eclipta prostrata L., is one of conventional Chinese medicine, there is nourishing
The effect of liver kidney, cooling blood and hemostasis.For the deficiency of liver-yin and kidney-yin, tooth mobility, poliosis, dizziness and tinnitus, soreness and weakness of waist and knees, YinXuXueRe
Haematemesis, bleeding from five sense organs or subcutaneous tissue, hematuria, bloody flux, metrostaxis, traumatic hemorrhage.Modern age pharmaceutical research shows that eclipta has immunological regulation, anti-ageing
Always, liver protection, the effect of antitumor isoreactivity.Chemical component contained by eclipta have triterpene saponin, thiophene-based, Coumarether,
Flavonoids etc.., with stronger clinical value, the market demand is wide for it, is developed to Chinese medicinal granule and related compound
Preparation is widely used in clinic.
The clinical use of Chinese medicine is mostly based on traditional decoction.The material base of traditional Chinese herbal decoction is prevented under instruction of Chinese Medicine theory
Control the basis of disease.Existing statutory standards carry out quantitative control only for single component, during dose-effect relationship cannot reflect comprehensively
The mass action of medicine ingredient.In the case where Chinese medicine overwhelming majority effective component at this stage is not known, traditional Chinese medicine fingerprint/spy
The foundation of sign map can greatly improve the technical level and scientific and technological content of traditional Chinese medicine quality control.
" Chinese Pharmacopoeia " version in 2015 using wedelolactone as eclipta quality evaluation index, only contains wedelolactone
Amount is controlled, and can not reflect its quality comprehensively.And the foundation about eclipta characteristic spectrum of literature research report at present,
Just for medicinal raw material, index components are mainly used for the Chinese medicine true and false, the place of production, product mostly using liposoluble constituent as research object
The Qualitive test of matter difference, it is more difficult to which the qualitative character of reflection eclipta comprehensively can not react the substance base of traditional Chinese medicine decoction
Plinth feature.
Summary of the invention
Based on this, the present invention provides the construction method and detection method of a kind of eclipta medicinal material UPLC characteristic spectrum.Building
Eclipta medicinal material characteristic spectrum have 8 water soluble ingredients characteristic peak, can realize quickly, comprehensively to eclipta medicinal material
The quality monitoring of multiple characteristic components, and the material base feature of the traditional Chinese medicine decoction of eclipta can be reacted.
The specific technical proposal is:
A kind of construction method of eclipta medicinal material UPLC characteristic spectrum, comprising the following steps:
The preparation of reference solution: respectively with wedelolactone and 4, bis--O- caffeoylquinic acids of 5- are reference substance, solubilization
Agent dissolution, prepares reference substance reference solution；Extraction solvent is added into eclipta control medicinal material, is heated to reflux, obtains extracting solution,
The extracting solution is filtered, takes subsequent filtrate as control medicinal material reference solution；
The preparation of test solution: being added Extraction solvent into eclipta medicinal material, be heated to reflux, and obtains extracting solution I, will be described
Extracting solution I filtration, takes subsequent filtrate as test solution I；Extraction solvent is added into eclipta standard decoction sample, heats back
Stream, obtains extracting solution II, and the extracting solution II is filtered, takes subsequent filtrate as test solution II；
Measurement: the reference substance reference solution, control medicinal material reference solution, test solution I and test sample is molten
It is measured in liquid II injection Ultra Performance Liquid Chromatography instrument, demarcates water-soluble shared peak to get eclipta medicinal material UPLC characteristic spectrum.
The chromatographic condition of the ultra performance liquid chromatography includes: in one of the embodiments,
Stationary phase: using octadecylsilane chemically bonded silica as the chromatographic column of filler；
Mobile phase: mobile phase A is acetonitrile, and Mobile phase B is triethylamine phosphate buffer, the triethylamine phosphate buffer with
Water is solvent, comprising volume fraction be 0.5%-0.6% phosphoric acid and volume fraction be 0.7%-0.83% triethylamine, gradient
Elution；
The gradient elution specifically:
0-3min, mobile phase A are increased to 16% by volume fraction for 11%, and Mobile phase B is 89% reduction by volume fraction
To 84%；
3min-9min, keeping mobile phase A volume fraction is 16%, and keeping Mobile phase B volume fraction is 84%；
9min-20min, mobile phase A are increased to 20% by volume fraction for 16%, and Mobile phase B is 84% by volume fraction
It is reduced to 80%；
20min-34min, keeping mobile phase A volume fraction is 20%, and keeping Mobile phase B volume fraction is 80%；
34min-40min, mobile phase A are increased to 80% by volume fraction for 20%, and Mobile phase B is 80% by volume fraction
It is reduced to 20%；
40min-41min, mobile phase A are reduced to 11% by volume fraction for 80%, and Mobile phase B is 20% by volume fraction
It is increased to 89%；
41min-50min, it is 11% that mobile phase A, which keeps volume fraction, and it is 89% that Mobile phase B, which keeps volume fraction,.
The chromatographic column is Agilent ZORBAX SBC18 in one of the embodiments,.
The chromatographic condition of the ultra performance liquid chromatography in one of the embodiments, further include:
28 DEG C -32 DEG C of column temperature, flow velocity is 0.28mL-0.32mL per minute, Detection wavelength 254nm-360nm.
The chromatographic condition of the ultra performance liquid chromatography in one of the embodiments, are as follows: Agilent ZORBAX SBC18
Chromatographic column (2.1 × 100mm, 1.8 μm)；Mobile phase A is acetonitrile, and Mobile phase B is triethylamine phosphate buffer, the triethylamine phosphorus
Acid buffer takes water as a solvent, comprising volume fraction be 0.53% phosphoric acid and volume fraction be 0.77%-0.83% three second
Amine, gradient elution, flow velocity: 0.3ml/min；Column temperature is 30 DEG C；Detection wavelength: 330nm.Number of theoretical plate is based on wedelolactone peak
8000 should be not less than by calculating.
The Extraction solvent is the methanol aqueous solution that volume fraction is 60%-80%, dosage in one of the embodiments,
50mL-100mL is added for every 1g eclipta medicinal material.
The time being heated to reflux is 60min-90min in one of the embodiments,.
The present invention also provides the detection methods of eclipta medicinal material.
The specific technical proposal is:
A kind of detection method of eclipta medicinal material, comprising the following steps:
The preparation of reference solution: respectively with wedelolactone and 4, bis--O- caffeoylquinic acids of 5- are reference substance, solubilization
Agent dissolution, prepares reference substance reference solution；Extraction solvent is added into eclipta control medicinal material, is heated to reflux, obtains extracting solution,
The extracting solution is filtered, takes subsequent filtrate as control medicinal material reference solution；
The preparation of testing sample solution: Extraction solvent being added into sample to be tested, is heated to reflux, and obtains extracting solution III, by institute
Extracting solution III filtration is stated, takes subsequent filtrate as testing sample solution；
Measurement: the reference substance reference solution, control medicinal material reference solution, testing sample solution are injected into ultra high efficiency
It is measured in liquid chromatograph.
In one of the embodiments, in the preparation of test solution, the eclipta medicinal material includes different sources
Eclipta medicinal material, test sample raw material of the invention come from the national biggish 6 genuine or major production areas of eclipta yield, and totally 18 batches
Secondary sample has adequately representative.
The chromatographic condition of the ultra performance liquid chromatography includes: in one of the embodiments,
Stationary phase: using octadecylsilane chemically bonded silica as the chromatographic column of filler；
Mobile phase: mobile phase A is acetonitrile, and Mobile phase B is triethylamine phosphate buffer, the triethylamine phosphate buffer with
Water is solvent, comprising volume fraction be 0.5%-0.6% phosphoric acid and volume fraction be 0.7%-0.83% triethylamine, gradient
Elution；
The gradient elution specifically:
0-3min, mobile phase A are increased to 16% by volume fraction for 11%, and Mobile phase B is 89% reduction by volume fraction
To 84%；
3min-9min, keeping mobile phase A volume fraction is 16%, and keeping Mobile phase B volume fraction is 84%；
9min-20min, mobile phase A are increased to 20% by volume fraction for 16%, and Mobile phase B is 84% by volume fraction
It is reduced to 80%；
20min-34min, keeping mobile phase A volume fraction is 20%, and keeping Mobile phase B volume fraction is 80%；
34min-40min, mobile phase A are increased to 80% by volume fraction for 20%, and Mobile phase B is 80% by volume fraction
It is reduced to 20%；
40min-41min, mobile phase A are reduced to 11% by volume fraction for 80%, and Mobile phase B is 20% by volume fraction
It is increased to 89%；
41min-50min, it is 11% that mobile phase A, which keeps volume fraction, and it is 89% that Mobile phase B, which keeps volume fraction,.
The chromatographic column is Agilent ZORBAX SBC18 in one of the embodiments,.
The chromatographic condition of the ultra performance liquid chromatography in one of the embodiments, further include:
28 DEG C -32 DEG C of column temperature, flow velocity is 0.28mL-0.32mL per minute, Detection wavelength 254nm-360nm.
The chromatographic condition of the ultra performance liquid chromatography in one of the embodiments, are as follows: Agilent ZORBAX SBC18
Chromatographic column (2.1 × 100mm, 1.8 μm)；Mobile phase A is acetonitrile, and Mobile phase B is triethylamine phosphate buffer, the triethylamine phosphorus
Acid buffer takes water as a solvent, comprising volume fraction be 0.53% phosphoric acid and volume fraction be 0.77%-0.83% three second
Amine, gradient elution, flow velocity: 0.3ml/min；Column temperature is 30 DEG C；Detection wavelength: 330nm.Number of theoretical plate is based on wedelolactone peak
8000 should be not less than by calculating.
The Extraction solvent is the methanol aqueous solution that volume fraction is 60%-80%, dosage in one of the embodiments,
50mL-100mL is added for every 1g eclipta medicinal material.
The time being heated to reflux is 60min-90min in one of the embodiments,.
Compared with prior art, the invention has the following advantages:
The present invention constructs the characteristic spectrum of eclipta medicinal material using ultra performance liquid chromatography (UPLC) method.Introduce eclipta mark
The characteristic spectrum of quasi- decoction is studied, the water soluble ingredient feature shared for eclipta standard decoction and eclipta medicinal material at
Divide to be studied and (8 common characteristic peaks is marked, and confirmed 5 common characteristic peaks using UPLC-MS and related control product
Ingredient, respectively caffeic acid, galuteolin, 3,4-Dicaffeoylquinic acid, 4,5-, bis--O- caffeoylquinic acids and wedelolactone) and conduct
The foundation that eclipta medicinal material characteristic spectrum characteristic peak determines can be good at characterizing the substance transmitting of medicinal raw material to decoction, no
It only can be good at control quality of medicinal material, and can also ensure that the quality of decoction；Meanwhile using reference substance, control medicinal material
Dual control, the durability offset issue that can effectively overcome liquid-phase condition finger-print to be inherently present compare more comprehensively.
The present invention is using assay peak (wedelolactone) as reference peak, it is specified that the opposite of other 7 characteristic peaks retains
Time and relative peak area limitation range, realize the quantification of multiple characteristic components, meet the mass action theory of Chinese medicine.
Present invention employs UPLC methods, compared with conventional H PLC method, more efficiently, quickly, environmental protection；It is constructed using the present invention
Characteristic spectrum and method, favorable reproducibility can accurately and reliably realize quickly, comprehensively to the multiple characteristic components of eclipta medicinal material
Quality monitoring, not only improved the quality control level of eclipta medicinal material, but also promote and stablize the inherent quality of eclipta medicinal material,
The raw material for meeting eclipta standard decoction requirement is provided for clinic, is provided for the relevant preparation process production process of eclipta important
Multi-index parameter foundation.
Detailed description of the invention
Fig. 1 is eclipta medicinal material characteristic spectrum under different flow phase systems；
Fig. 2 is eclipta medicinal material characteristic spectrum under different elution process；
Fig. 3 is eclipta medicinal material characteristic spectrum under different mobile phase buffers；
Fig. 4 is eclipta medicinal material characteristic spectrum under different wave length；
Fig. 5 is that integrality investigates characteristic spectrum；
Fig. 6 is the stacking chart (peak 1: caffeic acid of 18 batches of eclipta medicinal material characteristic spectrums；Peak 2: galuteolin；Peak 3: different green
Ortho acid B；Bis--O- caffeoylquinic acids of peak 5:4,5-；Peak 8 (s): wedelolactone)；
Fig. 7 is the stacking chart of 18 batches of eclipta standard decoction characteristic spectrums；
Fig. 8 is eclipta standard decoction characteristic spectrum；
Fig. 9 is eclipta mark medicinal material, eclipta standard decoction compare feature map；
Figure 10 is that specificity investigates characteristic spectrum；
Figure 11 is eclipta medicinal material compare feature map；
Figure 12 is sample to be tested characteristic spectrum.
Specific embodiment
Construction method and detection side below in conjunction with specific embodiment to eclipta medicinal material UPLC characteristic spectrum of the invention
Method is described in further detail.The invention can be realized in many different forms, however it is not limited to reality described herein
Apply mode.On the contrary, the purpose of providing these embodiments is that making to understand the disclosure of invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
The building of 1 eclipta medicinal material UPLC characteristic spectrum of embodiment
1, instrument and reagent
Water generation liquid chromatograph (model: H-Class；Detector: PDA；Producer: water generation instrument company)；Agilent
ZORBAX SBC18 chromatographic column (2.1 × 100mm, 1.8 μm of chromatographic column numbers: 037,025,040)；A ten thousandth balance
(ME204E, Mei Tele-support benefit Instrument Ltd.), hundred a ten thousandth balances (model: XP26, producer: Mei Tele-Tuo Li
Multiple instruments Co., Ltd)；Electric-heated thermostatic water bath (model: HWS28 type；Producer: the permanent Science and Technology Ltd. in Shanghai one)；Numerical control is super
Sound wave washer (model: KQ-500DE；Producer: Kunshan Ultrasonic Instruments Co., Ltd.)；Ultrapure water system (model: Milli-
Q-Direc；Producer: Merck S. A.).
Methanol, ethyl alcohol are to analyze pure (Tianjin Fu Yu Fine Chemical Co., Ltd), and liquid phase methanol, acetonitrile are chromatographically pure
(Merck S. A.), triethylamine, phosphoric acid are chromatographically pure (Tianjin Kermel Chemical Reagent Co., Ltd.), and water is ultrapure
Water (is derived from laboratory Milli-Q ultrapure water system, Merck S. A.).
2, reagent
Wedelolactone reference substance (lot number: 111885-201403, in terms of 99.6%, grind content by Chinese food drug assay
Study carefully institute)；(lot number: 111894-201102, content is in terms of 94.10%, Chinese food for 4,5- bis--O- caffeoylquinic acids reference substances
Drug assay research institute)；Eclipta medicinal material (18 batches of eclipta medicinal material information are shown in Table 2-2).
Crude drug source: this research has collected totally 18 batches of eclipta crude drugs altogether, as shown in table 1, wherein coming from Guangdong Province 3
Batch, Shandong Province 3 batches, 6 batches, Jiangsu Province, 3 batches, Anhui Province, 2 batches, Henan Province, Guangxi province 1 batch.Detailed derivation: slightly.
Table 1
Remarks: wedelolactone content is by the lower content assaying method measurement of Chinese Pharmacopoeia (2015 editions) eclipta.
3, the preparation of reference solution
(1) preparation of reference substance reference solution:
Wedelolactone reference substance 3.868mg is taken, is set in 10ml volumetric flask, adds methanol that every 1ml is made containing wedelolactone
The solution of 385.2528 μ g draws above-mentioned 200 μ l of mother liquor, sets in 10ml volumetric flask, 70% ethyl alcohol is added to be made often to get mother liquor
The solution of the 1ml 7.7051 μ g containing wedelolactone respectively is to get wedelolactone reference substance reference solution.
4,5-, bis--O- caffeoylquinic acids reference substance 2.143mg is taken, sets in 10ml volumetric flask, adds 70% (volume fraction)
Ethanol water be made the solution of every 1ml 201.6563 μ g containing wedelolactone to get.Above-mentioned mother liquor is respectively drawn 200 μ l and is set
In 10ml volumetric flask, adds 70% (volume fraction) ethanol water that every 1ml is made and contain 7.7051 μ g of wedelolactone respectively, 4,5-
The solution of two-O- caffeoylquinic acids 4.0331ug is to get 4,5-, bis--O- caffeoylquinic acids reference substance reference solution.
(2) control medicinal material reference solution: taking eclipta control medicinal material powder (crossing No. three sieves) 1g, accurately weighed, sets tool
It filling in conical flask, 70% (volume fraction) ethanol water 50ml is added in precision, and weighed weight is heated to reflux 1h, takes extracting solution,
The extracting solution is let cool, the weight of less loss is supplied with 70% (volume fraction) ethanol water, constant volume shakes up, and filtration takes continuous
Filtrate is to get control medicinal material reference solution.
(3) preparation of standard decoction: taking eclipta medicine materical crude slice 100g, adds water to cook secondary, first time decoction plus 14 times of amount water,
It impregnates 30 minutes, changes mild fire after boiling by intense fire and decoct again 60 minutes, filtered while hot with 350 meshes, the rapid cold water of filtrate is cooling；The
Secondary decoct adds 11 times of amount water, changes mild fire after boiling by intense fire and decocts again 40 minutes, is filtered while hot with 350 meshes, filtrate is cold rapidly
Water is cooling；Merge filtrate twice, is concentrated under reduced pressure into after clear cream solid content is about 12%, dispenses into cillin bottle, every bottle of packing
2ml, vacuum freeze drying are taken out, roll aluminium lid to obtain the final product.
4, the preparation of test solution
(1) preparation of test solution: taking eclipta medicinal powder (crossing No. three sieves) 0.2g, accurately weighed, sets tool plug cone
In shape bottle, 70% (volume fraction) ethanol water 50ml is added in precision, and weighed weight is heated to reflux 1h, takes extracting solution, by institute
It states extracting solution to let cool, the weight of less loss is supplied with 70% (volume fraction) ethanol water, constant volume shakes up, and filtration takes continuous filter
Liquid is to get test solution.
(2) preparation of standard decoction test solution
Eclipta standard decoction sample about 0.2g is taken, it is accurately weighed, it sets in stuffed conical flask, 70% (volume is added in precision
Score) ethyl alcohol 25ml, weighed weight is heated to reflux 30 minutes, lets cool, and the weight of less loss is supplied with 70% (volume fraction) ethyl alcohol
Amount, shake up, filter, take subsequent filtrate to get.
5, the determination of chromatographic condition
(2) flow phase system selects
Investigate 4 kinds of different flow phase systems, methanol-water, acetonitrile-water, methanol -0.2% (volume fraction) phosphate aqueous solution,
Acetonitrile -0.2% (volume fraction) phosphate aqueous solution, selects optimal flow phase system as the mobile phase of eclipta characteristic spectrum
System.
Chromatographic condition: Agilent SB C18 (2.1mm × 100mm, 1.8 μm)；Column temperature: 30 DEG C；Sample volume: 1 μ l；Detection
Wavelength: 330nm；Using acetonitrile/methanol as mobile phase A, using 0.2% phosphoric acid/water as Mobile phase B, gradient condition: 0~30min:5%
→ 95%；Flow velocity is 0.3ml/min.The result is shown in Figure 1.
By comparing methanol-water, methanol-acid, acetonitrile-water, acetonitrile-acid system map, acetonitrile-sour water system diagram is found
Spectrum color spectral peak information is relatively abundanter, and peak type is sharp, symmetrical, therefore uses acetonitrile-sour water system.
(3) gradient method is investigated
4 kinds of gradient methods in table 2-5 are compared, optimal conduct eclipta characteristic spectrum elution process is chosen.
Chromatographic condition: Agilent SB C18 (2.1mm × 100mm, 1.8 μm)；Column temperature: 30 DEG C；Sample volume: 1 μ l；Detection
Wavelength: 330nm；Using acetonitrile as mobile phase A, using 0.2% phosphate aqueous solution as Mobile phase B, by gradient specified in table 2~5 into
Row elution, flow velocity 0.3ml/min.As a result see Fig. 2.
Table 2
Table 3
Table 4
Table 5
It compared 4 kinds of gradients, wherein the separating effect of method 4 and peak type are preferable, and analysis time is shorter, therefore use
Method 4 is used as final gradient.
(4) mobile phase is investigated
0.2% (volume fraction) aqueous acetic acid, 0.2% (volume fraction) aqueous formic acid, 0.2% are chosen in this experiment
(volume fraction) phosphate aqueous solution, triethylamine phosphate buffer (take water as a solvent, comprising volume fraction be 0.53% phosphoric acid and
Volume fraction is 0.8% triethylamine, PH2.4), 4 kinds of mobile phases are compared, and determine suitable mobile phase.
Chromatographic condition: Agilent SB C18 (2.1mm × 100mm, 1.8 μm)；Column temperature: 30 DEG C；Sample volume: 1 μ l；Detection
Wavelength: 330nm；Using acetonitrile as mobile phase A, respectively with 0.2% phosphoric acid, 0.2% acetic acid, 0.2% formic acid, triethylamine phosphoric acid buffer
Liquid is Mobile phase B, is eluted by gradient specified in table 5, flow velocity 0.3ml/min.As a result see Fig. 3.
Compare 4 kinds of different mobile phase acidity, different mobile phase acidity are affected to peak type and appearance time, triethylamine phosphorus
Acid buffer (PH2.4) system effect is preferable, and the separating effect and peak type of each chromatographic peak reach requirement, final to determine triethylamine phosphorus
Acid buffer (PH=2.4) is used as mobile phase.
(5) determination of optimal absorption wavelength
By consulting literatures it is found that the measurement wavelength of eclipta characteristic spectrum mainly has 270nm, 330nm.Two kinds of waves herein
It on the basis of length, is compared in conjunction with 254nm, 300nm, 360nm, determines optimal absorption wavelength.
Chromatographic condition: Agilent SB C18 (2.1mm × 100mm, 1.8 μm)；Column temperature: 30 DEG C；Sample volume: 1 μ l；Detection
Wavelength: 254nm, 270nm, 300nm, 330nm, 360nm；Using acetonitrile as mobile phase A, triethylamine phosphate buffer (is molten with water
Agent, comprising volume fraction be 0.53% phosphoric acid and volume fraction be 0.8% triethylamine, PH2.4) be Mobile phase B, by table 5
Defined gradient is eluted, flow velocity 0.3ml/min.As a result see Fig. 4.
Experimental result: more several difference channels find that the peak information of 330nm is more complete, and each main peaks absorb by force, therefore select
330nm is as Detection wavelength.
(6) integrity analysis
Integrity analysis is carried out to above-mentioned chromatographic condition, investigates whether sample elutes completely.
Chromatographic condition: Agilent SB C18 (2.1mm × 100mm, 1.8 μm)；Column temperature: 30 DEG C；Sample volume: 1 μ l；Detection
Wavelength: 330nm；Using acetonitrile as mobile phase A, with triethylamine phosphate buffer (PH2.4) for Mobile phase B, by ladder specified in table 5
Degree is eluted, flow velocity 0.3ml/min.As a result see Fig. 5.
The results showed that without obvious chromatographic peak after 45 minutes, which can be by ingredient most in eclipta
It separates, will not influence next needle.
(7) determination of chromatographic condition
Chromatographic condition: Agilent SB C18 (2.1 × 100mm, 1.8 μm)；Column temperature: 30 DEG C；Sample volume: 1 μ l；Detect wave
It is long: 330nm；Using acetonitrile as mobile phase A, (takes water as a solvent with triethylamine phosphate buffer, be 0.53% comprising volume fraction
Phosphoric acid and volume fraction are 0.8% triethylamine, PH2.4) it is Mobile phase B, gradient condition such as table 5；Flow velocity is 0.3ml/min.
6, sample solution preparation method is investigated
To the Extraction solvent of eclipta medicinal material sample solution preparation method, extracting mode, solvent usage, extraction time into
Row is investigated, and determines sample solution preparation method
(1) Extraction solvent is investigated
This is tested has investigated influence of the different solvents to eclipta medicinal material characteristic spectrum respectively, selection methanol,
70% (volume fraction) methanol aqueous solution, ethyl alcohol, 70% (volume fraction) ethanol water, Diluted Alcohol are right as Extraction solvent
The sample solution characteristic spectrum of different solvents is measured, and determines optimum extraction solvent.
The preparation of test solution: taking eclipta medicinal material (lot number: G1705159) 1.0g, parallel 5 parts, accurately weighed, sets
It is accurate respectively that methanol, 70% methanol, ethyl alcohol, 70% ethyl alcohol, Diluted Alcohol 50ml, weighed weight, heating is added in stuffed conical flask
Flow back 1h, extracting solution taking-up is let cool, then weighed weight, supplies less loss weight with corresponding solvent respectively, shakes up, filter to get.
By the progress of 5 (7) chromatographic conditions, sample introduction is analyzed, and the investigation of eclipta medicinal material characteristic spectrum different solvents the results are shown in Table
6。
Table 6
By compare 5 kinds of different solvents chromatogram can find, the chromatographic peak number of 5 kinds of Extraction solvent chromatograms with
Chromatographic peak peak shape is consistent, the sequence of total peak area size are as follows: Diluted Alcohol > 70% ethyl alcohol > 70% methanol > ethyl alcohol > methanol；Synthesis is examined
Consider extractability, chromatographic peak peak shape, peak area/sample weighting amount value, solvent effect and the toxicity size etc. of each solvent, it is final to use
70% (volume fraction) ethanol water is as Extraction solvent.
(2) extracting mode is investigated
Influence of the different extracting modes to eclipta medicinal material characteristic spectrum is investigated in this experiment, chooses refluxing extraction and ultrasound
Extract two ways.The sample solution characteristic spectrum of different extracting modes is measured, determines optimum extraction mode.
The preparation of test sample: taking eclipta mark medicinal material (lot number: G1705159) 1.0g, parallel 2 parts, accurately weighed, sets tool
Fill in conical flask, accurate respectively to be added 70% ethyl alcohol 50ml, weighed weight, be respectively adopted refluxing extraction 1h, ultrasound (250W,
40KHz) 1h respectively lets cool extracting solution, and the weight of less loss is supplied with 70% ethyl alcohol, is shaken up, filtration, take subsequent filtrate to get.
By the chromatographic condition progress of 5 (7), sample introduction is analyzed.Eclipta medicinal material characteristic spectrum difference extracting mode is investigated result and is seen
Table 7.
Table 7
It compared two kinds of extracting modes, the chromatogram chromatographic peak number of different extracting modes is consistent with chromatographic peak peak shape, peak
Area adduction/sample weighting amount sequence are as follows: reflux > ultrasound, therefore select reflux as processing mode.
(3) solvent usage is investigated
Influence of the different solvents dosage to eclipta medicinal material characteristic spectrum is investigated in this experiment, is chosen solvent usage and is
15ml、25ml、50ml、100ml。
Eclipta medicinal powder (crossing No. three sieves) (lot number: G1705159) 1.0g is taken, it is parallel 4 parts, accurately weighed, set tool plug
In conical flask, difference 70% ethyl alcohol 15ml, 25ml, 50ml, 100ml of accurate addition, weighed weight, heating and refluxing extraction 1 hour,
Extracting solution is let cool, then weighed weight, supplies less loss weight with 70% ethyl alcohol, shake up, filter, take subsequent filtrate to get.
By the progress of 5 (7) chromatographic conditions, sample introduction is analyzed.The investigation of eclipta medicinal material characteristic spectrum different solvents dosage the results are shown in Table
8。
Table 8
Experimental result: influence of the different solvents dosage to eclipta medicinal material characteristic spectrum, total peak area size be compared
Sequential bits 50ml > 100ml > 25ml > 15ml, the main peaks responsiveness of 100ml is smaller, from " peak area adduction × extension rate/
From the aspect of sample weighting amount " result and saving solvent, consider in conjunction with solvent is saved, solvent usage selects 50ml.
(4) extraction time is investigated
Influence of the different extraction times to eclipta medicinal material characteristic spectrum is investigated in this experiment, chooses the ultrasonic extraction time point
Not are as follows: 15min, 30min, 60min, 90min.The sample solution characteristic spectrum of different extraction times is measured, is determined most
Good extraction time.
The preparation of test sample: taking eclipta mark medicinal material (lot number: G1705159) about 1.0g, accurately weighed, sets tool plug taper
It is accurate respectively to be added 70% ethyl alcohol 50ml in bottle, weighed weight, be respectively adopted refluxing extraction 15min, 30min, 60min,
90min lets cool extracting solution, and the weight of less loss is supplied with 70% ethyl alcohol, is shaken up, filtration, take subsequent filtrate to get.
By the chromatographic condition progress of 5 (7), sample introduction is analyzed.Eclipta medicinal material characteristic spectrum difference extracting mode is investigated result and is seen
Table 9.
Table 9
By the eclipta medicinal material characteristic spectrum for comparing different return times, it is possible to find with the increase of return time, color
Spectrogram total peak area is increased slightly, and reflux 60min and 90min peak area difference is unobvious, and the 60min that illustrates to flow back is black drought
The main component of lotus is extracted completely substantially, and choosing return time for convenience of experiment is 60min.
(5) preparation method of test solution determines
By being investigated to different solvents, solvent usage, different extracting modes and different extraction times, determines and supply
Test product the preparation method comprises the following steps: take eclipta medicinal powder (cross No. three sieve) 1.0g, it is accurately weighed, set in stuffed conical flask, it is accurate
70% ethyl alcohol 50ml is added, weighed weight is heated to reflux 1 hour, extracting solution is let cool, then weighed weight, is mended with 70% ethyl alcohol
Sufficient less loss weight, shakes up, and filtration takes subsequent filtrate to get test solution.
7, eclipta medicinal material characteristic spectrum shares the determination at peak
18 batches of eclipta medicinal materials are taken, according to the preparation method of test solution in 4 (1), 18 crowdes of test solution I are made,
Above-mentioned 18 crowdes of test solution I are injected in liquid chromatograph, using the chromatographic condition in 5 (7), sample introduction measurement obtains 18 batches
Eclipta medicinal material characteristic spectrum.Referring to Fig. 6.
18 batches of eclipta standard decoction samples separately are taken, according to the preparation method of 4 (2) Plays decoction test solutions, system
18 crowdes of test solution II are obtained, above-mentioned 18 crowdes of test solution II are injected in liquid chromatograph, using the chromatostrip in 5 (7)
Part, sample introduction measurement, obtains 18 batches of eclipta standard decoction sample characteristic maps.Referring to Fig. 7.
The experimental results showed that 18 batches of eclipta medicinal material characteristic spectrums have 8 characteristic peaks, and the equal energy of 8 characteristic peaks
It is transferred in eclipta standard decoction from eclipta medicinal material stabilization, i.e. eclipta medicinal material characteristic spectrum and standard decoction characteristic spectrum
In 8 characteristic peaks it is corresponding, therefore, select this 8 characteristic peaks for the shared peak of eclipta medicinal material, and with peak 8 (in wedelia chinensis
Ester) it is to carry out research on standard referring to peak, details are referring to Fig. 8, Fig. 9.
8, eclipta medicinal material characteristic spectrum methodology validation
(1) specificity is investigated
Take test solution under eclipta (G1705159) characteristic pattern spectral term, blank solvent (70% ethyl alcohol), object of reference molten
Liquid is measured by the chromatographic condition sample introduction established, and is recorded chromatogram (Figure 10).
Experimental result shows 8 chromatographic peaks of eclipta medicinal material characteristic spectrum not by the interference of negative blank solvent, wherein 4,
Bis--O- caffeoylquinic acids of 5-, wedelolactone chromatographic peak are consistent with the retention time of reference solution, and this method has good
Specificity.
(2) precision is investigated
By the test solution of preparation method preparation eclipta (G1705159) of test solution in 4 (1), precision is drawn
The test solution of eclipta medicinal material is measured, with wedelia chinensis by 6 injection UPLC of chromatographic condition continuous sample introduction in 5 (7)
Lactone chromatographic peak is to calculate the relative retention time and relative peak area at 1~peak of peak 8 referring to peak S；And RSD value is calculated, it tests
It the results are shown in Table 10 and table 11.
10 eclipta medicinal material characteristic spectrum precision of table investigates result (relative retention time)
11 eclipta medicinal material characteristic spectrum precision of table investigates result (relative peak area)
Experimental result shows that each chromatographic peak relative peak area and relative retention time RSD are in 0.03%~1.75% range
It is interior, show that instrument precision is good.
(3) repeatability is investigated
By the test solution of preparation method preparation eclipta (G1705159) of test solution in 4 (1), precision is drawn
The test solution of eclipta medicinal material, parallel 6 parts, by the chromatographic condition in 5 (7), sample introduction measurement, with wedelolactone chromatographic peak
To calculate the relative retention time and relative peak area at 1~peak of peak 8 referring to peak S；And calculate RSD value.It the results are shown in Table 12 and table
13。
12 eclipta medicinal material characteristic spectrum repeatability of table investigates result (relative retention time)
13 eclipta medicinal material characteristic spectrum repeatability of table investigates result (relative peak area)
Experimental result shows that 6 parts of samples of replication, each chromatographic peak relative retention time RSD is 0.02%~0.19%
In range, relative peak area RSD shows that this feature atlas analysis method repeatability is good in 0.83%~1.78% range.
(4) study on the stability
By the test solution of preparation method preparation eclipta (G1705159) of test solution in 4 (1), at room temperature
It places, precision draws the test solution of eclipta medicinal material, by the chromatographic condition in 5 (7) 0 hour, 2 hours, 6 hours, 12
The sample introduction measurement of hour, 18 hours, 24 hours is to calculate the reservation relatively at 1~peak of peak 4 referring to peak S with wedelolactone chromatographic peak
Time and relative peak area；And calculate RSD value.It the results are shown in Table 14 and table 15.
14 eclipta medicinal material characteristic spectrum study on the stability result (relative retention time) of table
15 eclipta medicinal material characteristic spectrum study on the stability result (relative peak area) of table
Experimental result shows, each chromatographic peak relative retention time RSD of 24 hours test solutions 0.10%~
0.19%, relative peak area shows that test solution is stablized in 24 hours in 0.34%~5.97% range.
(5) Intermediate precision is investigated
Eclipta is prepared by the preparation method of test solution in 4 (1) in not same date by other analysis personnel
(G1705159) test solution, the test solution of precision absorption eclipta medicinal material, parallel 6 parts, and in different chromatographs
Lower operation, by the chromatographic condition in 5 (7), sample introduction is analyzed, is to calculate 1~peak of peak 8 referring to peak S with wedelolactone chromatographic peak
Relative retention time and relative peak area；And compared with repeatability investigation data, RSD value is calculated.It the results are shown in Table 16 and table 17.
16 eclipta medicinal material characteristic spectrum Intermediate precision of table investigates result (relative retention time)
17 eclipta medicinal material characteristic spectrum Intermediate precision of table investigates result (relative peak area)
Experimental result shows that different analysis personnel operate under not same date and different chromatographs, relative retention time RSD
In 0.03%~0.19% range, chromatographic peak relative peak area RSD shows each chromatographic peak in 0.55%~1.91% range
Relative retention time Intermediate precision is preferable.
(6) durability is investigated
1. different column temperatures are investigated
Compare different column temperatures, being respectively 28 DEG C, 30 DEG C, 32 DEG C influences eclipta medicinal material characteristic spectrum.
By the test solution of preparation method preparation eclipta (G1705159) of test solution in 4 (1), precision is drawn
The test solution of eclipta medicinal material, by the chromatographic condition in 5 (7), (column temperature is changed to 28 DEG C, 30 DEG C, 32 DEG C respectively), sample introduction is surveyed
It is fixed, it is to calculate relative retention time and relative peak area referring to peak with No. 8 peaks wedelolactone peak.It is shown in Table 18 and table 19.
18 eclipta medicinal material column temperature of table investigates result (relative retention time)
19 eclipta medicinal material column temperature of table investigates result (relative peak area)
Experimental result shows, when different column temperatures, each chromatographic peak relative retention time RSD is in 0.08%~2.36% range
Interior, relative peak area RSD illustrates that this method is good in column temperature amplitude of variation hour durability in 0.88%~5.25% range
It is good.
2. investigation different in flow rate
It is more different in flow rate, it is 0.28ml/min, 0.30ml/min, 0.32ml/min respectively to eclipta medicinal material characteristic pattern
Composing durability influences.
By the test solution of preparation method preparation eclipta (G1705159) of test solution in 4 (1), precision is drawn
The test solution of eclipta medicinal material, by 5 (7) chromatographic condition (flow velocity be changed to respectively 0.28ml/min, 0.30ml/min,
0.32ml/min), sample introduction measures, and is to calculate relative retention time and opposite peak face referring to peak with No. 8 peaks wedelolactone peak
Product.It the results are shown in Table 20 and table 21.
20 eclipta medicinal material flow velocity of table investigates result (relative retention time)
21 eclipta medicinal material flow velocity of table investigates result (relative peak area)
Experimental result is shown, when different in flow rate, each chromatographic peak relative retention time RSD in 0.2%~2.47% range,
Relative peak area RSD shows that analysis method durability different in flow rate is preferable in 2.37%~3.35% range.
3. different chromatographic columns are investigated
Compare Agilent SB C18 chromatographic column (number 025,037,040) to eclipta medicinal material characteristic spectrum durability
It influences.
By the test solution of preparation method preparation eclipta (G1705159) of test solution in 4 (1), precision is drawn
The test solution of eclipta medicinal material (uses Agilent SB C18 chromatographic column, number is by the chromatographic condition in 5 (7) respectively
025,037,040), sample introduction measurement is to calculate relative retention time and opposite peak face referring to peak with No. 8 peaks wedelolactone peak
Product.It the results are shown in Table 22 and table 23.
22 eclipta medicinal material characteristic spectrum chromatographic column of table investigates result (relative retention time)
23 eclipta medicinal material characteristic spectrum chromatographic column of table investigates result (relative peak area)
Experimental result is shown, when fixing Agilent SB C18 chromatographic column, investigates the chromatographic column of different batches, each chromatographic peak
Relative retention time RSD is in 0.31%~3.66% range, in addition to peak 4, the relative peak area RSD at other peaks 1.53%~
In 3.32% range, show that the analysis method difference chromatographic column durability is preferable.Since peak 4 is by the separating effect shadow of chromatographic column
It rings, separating degree is bad, therefore suggests that the relative peak area at peak 4 should not be defined.
4. different acidity buffer is investigated
Compare influence of the phosphoric acid triethylamine buffer of different acidity to eclipta medicinal material characteristic spectrum.Three kinds of buffers
Preparation method is as follows:
(1) triethylamine phosphate buffer (12:8) [12ml triethylamine adds 8ml phosphoric acid to be settled to 1000ml, then plus 500ml it is dilute
It releases.It is equivalent to and takes water as a solvent, contains 0.53% (volume fraction) phosphoric acid and 0.8% (volume fraction) triethylamine]: ginseng
The preparation method of 2015 editions the 4th middle triethylamine buffer solutions of Chinese Pharmacopoeia is examined, the configuration method of this buffer is to pipette phosphoric acid
8ml, triethylamine 12ml are set in 1000ml volumetric flask, and water is added and is settled to 1000ml, shakes up, pours out and set in beaker, water is added
500ml is stirred evenly.Later period for convenience of configuring, is changed to pipette 5.3ml phosphoric acid and 8ml triethylamine, it is mixed that 1000ml water is added
It is even can to get arrive triethylamine phosphate buffer.
(2) triethylamine phosphate buffer (11.5:8) [is equivalent to and takes water as a solvent, contain 0.53% (volume fraction) phosphoric acid
With 0.77% (volume fraction) triethylamine]: it takes phosphoric acid 8ml, triethylamine 11.5ml to set in 1000ml volumetric flask, water is added and is settled to
1000ml shakes up, and pours out and sets in beaker, and water 500ml is added, stirs evenly.
(3) triethylamine phosphate buffer (12.5:8) [is equivalent to and takes water as a solvent, contain 0.53% (volume fraction) phosphoric acid
With 0.83% (volume fraction) triethylamine]: it takes phosphoric acid 8ml, triethylamine 12.5ml to set in 1000ml volumetric flask, water is added and is settled to
1000ml shakes up, and pours out and sets in beaker, and water 500ml is added, stirs evenly.
By the test solution of preparation method preparation eclipta (G1705159) of test solution in 4 (1), precision is drawn
The test solution of eclipta medicinal material, by the chromatographic condition in 5 (7), sample introduction measurement.Using No. 8 peak wedelolactones as reference, meter
Relative retention time and relative peak area are calculated, experimental result is shown in charts 24, table 25.
24 eclipta medicinal material buffer salt of table investigates result (relative retention time)
25 eclipta medicinal material buffer salt of table investigates result (relative peak area)
Experimental result is shown: each chromatographic peak relative retention time RSD is 0.06%~0.66% under different buffer conditions
In range, relative peak area RSD shows that this method is resistance to the smaller variation of buffer salt acidity in 1.42%~4.24% range
It is preferable with property.
In conclusion providing its determination value in eclipta medicinal material standard, relative retention time is relatively stable, column temperature, flow velocity,
The small variations of buffer salt ratio are smaller on the influence of the measurement result of eclipta medicinal material characteristic spectrum, can carry out in the appropriate range
Adjustment.
9, the determination of eclipta medicinal material characteristic spectrum method
It can determine eclipta medicinal material characteristic spectrum measuring method according to above-mentioned experimental result are as follows:
Chromatographic condition and system suitability: Agilent ZORBAX SBC18 chromatographic column (2.1 × 100mm, 1.8 μm)；
Using acetonitrile as mobile phase A, (taken water as a solvent, the phosphoric acid and body for being 0.53% comprising volume fraction with triethylamine phosphate buffer
Fraction is 0.8% triethylamine, PH2.4) it is Mobile phase B, it provides to carry out gradient elution by table 5；Flow velocity: 0.3ml/min；Column temperature
It is 30 DEG C；Detection wavelength: 330nm.Number of theoretical plate is calculated by wedelolactone peak should be not less than 8000.
The preparation of reference solution: taking wedelolactone and 4,5-Dicaffeoylquinic acid reference substance appropriate, accurately weighed, adds 70% second
Alcohol is made every 1ml and contains the mixed solution of 8 μ g of wedelolactone, 4,5-, bis--O- caffeoylquinic acids, 4 μ g to get as reference substance
Reference solution；Eclipta control medicinal material powder (crossing No. three sieves) 1g is separately taken, it is accurately weighed, it sets in stuffed conical flask, precision adds
Entering 70% (volume fraction) ethanol water 50ml, weighed weight is heated to reflux 1h, takes extracting solution, the extracting solution is let cool,
The weight of less loss is supplied with 70% (volume fraction) ethanol water, constant volume shakes up, and filtration takes subsequent filtrate to get control medicinal material
Reference solution.
The preparation of test solution: taking eclipta medicinal powder (crossing No. three sieves) 1g, accurately weighed, sets stuffed conical flask
In, 70% ethyl alcohol 50ml is added in precision, and weighed weight is heated to reflux 1h, extracting solution is let cool, and supplies less loss with 70% ethyl alcohol
Weight shakes up, filtration, take subsequent filtrate to get.
Measuring method: it is accurate respectively to draw reference solution and each 1 μ l of test solution, liquid chromatograph is injected, is measured, i.e.,
?.
10, the determination of eclipta medicinal material characteristic spectrum
(1) 18 batches of eclipta medicinal material characteristic spectrums are analyzed, is to calculate 8 referring to peak with wedelolactone chromatographic peak
Result table 26, table 27 are shown in the relative retention time and relative peak area of a chromatographic peak, experiment:
26 eclipta medicinal material sample measurement result (relative retention time) of table
27 eclipta medicinal material sample measurement result (relative peak area) of table
The results show that 18 batches of eclipta medicinal material characteristic spectrums have 8 shared peaks, with eclipta standard decoction characteristic spectrum
Shared peak is consistent.
It is that remaining 8 characteristic peak of 18 batches of eclipta medicinal material characteristic spectrums are opposite referring to peak with 8 wedelolactone of peak to retain
Time RSD value meets the standard requirements of eclipta medicinal material characteristic spectrum 0.14%~1.66%, respectively less than 2.0%；18 batches of ink
The RSD of 8 characteristic peak relative peak areas of non-irrigated lotus medicinal material characteristic spectrum is 42.19%~102.16%, the results showed that difference batch
There is some difference for the characteristic peak tie element of secondary eclipta medicinal material.In conjunction with above-mentioned relative peak area, batch is G1710015's
The peak 1,2,4,5,6,7 of sample differs greatly with the relative peak area of other batches, belongs to Outlier Data, therefore this batch is deleted
It goes to determine peak area range again；The relative peak area at each peak G1705160 is integrally less than normal, therefore rejects.Therefore 1~peak of peak 7 is with respect to peak
Areal extent is successively are as follows: 0.008~0.129,0.1030~0.3472,0.0593~0.630,0.159~1.339,0.146~
1.893,0.155~0.819,0.554~1.352.
For the quality of strict control eclipta medicinal material, for eclipta standard decoction and related preparations preparation provide it is of fine quality and
Stable raw medicinal material sets limit standard to the characteristic peak relative peak area of eclipta medicinal material characteristic spectrum, is very necessary
's.
(2) characteristic spectrum is drafted
" chromatographic fingerprints of Chinese materia medica similarity evaluation software systems " are used to match 18 batches of eclipta medicinal material characteristic spectrums,
Control map is generated, eclipta medicinal material compare feature map is established, as a result such as Figure 11.Compare the following table 28 of profile information.
Table 28
Tentative eclipta medicinal material characteristic spectrum standard are as follows: 8 characteristic peaks should be presented in sample chromatogram, with wedelolactone
It is the peak S referring to the corresponding peak 8 in peak, calculates the relative retention time of each characteristic peak Yu the peak S, relative retention time should be in specified value
± 10% within, it is specified that value are as follows: 0.13 (peak 1), 0.28 (peak 2), 0.37 (peak 3), 0.43 (peak 4), 0.73 (peak 6), 0.79
(peak 7)；The relative peak area of each characteristic peak Yu the peak S is calculated, relative peak area should be within the specified scope, it is specified that range are as follows:
0.008~0.129 (peak 1), 0.1030~0.3472 (peak 2), 0.0591~0.630 (peak 3), 0.159~1.339 (peak 4),
0.146~1.893 (peak 5), 0.395~0.819 (peak 6), 0.554~1.352 (peak 7).
11, eclipta standard decoction chromatographic peak points out research
The material composition that may contain in eclipta standard decoction is confirmed using LC-MS technology by literature research,
Again by the reference substance that can be obtained, pointed out, as a result as follows:
5 shared peaks in 8 characteristic peaks of eclipta standard decoction are pointed out.Being respectively as follows: shared peak peak 1 is caffeic acid；
Peak 2 is galuteolin；Peak 3 is 3,4-Dicaffeoylquinic acid；Peak 5 is 4,5-Dicaffeoylquinic acid；Peak 8 is wedelolactone.
The detection method of 2 eclipta medicinal material of embodiment
The present embodiment provides a kind of identification applications of eclipta medicinal material, and steps are as follows:
(1) chromatographic condition: with 5 (7) in embodiment 1.
(2) preparation of reference solution: with 4 in embodiment 1.
(3) preparation of testing sample solution: taking sample to be tested (crossing No. three sieves) 1g, accurately weighed, sets in stuffed conical flask,
70% (volume fraction) ethanol water 50ml is added in precision, and weighed weight is heated to reflux 1h, takes extracting solution, by the extraction
Liquid is let cool, and the weight of less loss is supplied with 70% (volume fraction) ethanol water, and constant volume shakes up, filtration, take subsequent filtrate to get
Testing sample solution.
(4) measure: precision draws above-mentioned reference solution, testing sample solution, injects in liquid chromatograph, and measurement obtains
To characteristic spectrum, as shown in figure 12.
29 sample to be tested characteristic spectrum relative retention time of table and relative peak area
The eclipta medicinal material characteristic spectrum (Figure 12) to be measured is compared with eclipta medicinal material compare feature map (Figure 11)
Compared with: data result shows (table 23), which can detect 8 characteristic peaks, and with 8 in compare feature map
Characteristic peak is corresponding；Its relative peak area and relative retention time in the range of standard provides, thus illustrate, batch ink
Non-irrigated lotus quality of medicinal material is qualified, and quality is more stable, meets the requirement of clinical decoction.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of construction method of eclipta medicinal material UPLC characteristic spectrum, which comprises the following steps:
The preparation of reference solution: respectively with wedelolactone and 4, bis--O- caffeoylquinic acids of 5- are reference substance, and solubilizer is molten
Solution, prepares reference substance reference solution；Extraction solvent is added into eclipta control medicinal material, is heated to reflux, obtains extracting solution, by institute
Extracting solution filtration is stated, takes subsequent filtrate as control medicinal material reference solution；
The preparation of test solution: being added Extraction solvent into eclipta medicinal material, be heated to reflux, and obtains extracting solution I, by the extraction
Liquid I filtration, takes subsequent filtrate as test solution I；Extraction solvent is added into eclipta standard decoction sample, is heated to reflux,
Extracting solution II is obtained, the extracting solution II is filtered, takes subsequent filtrate as test solution II；
Measurement: by the reference substance reference solution, control medicinal material reference solution, test solution I and test solution II
It is measured in injection Ultra Performance Liquid Chromatography instrument, demarcates water-soluble shared peak to get eclipta medicinal material UPLC characteristic spectrum.
2. construction method according to claim 1, which is characterized in that the chromatographic condition packet of the ultra performance liquid chromatography
It includes:
Stationary phase: using octadecylsilane chemically bonded silica as the chromatographic column of filler；
Mobile phase: mobile phase A is acetonitrile, and Mobile phase B is triethylamine phosphate buffer, and the triethylamine phosphate buffer is with water
Solvent, comprising volume fraction be 0.5%-0.6% phosphoric acid and volume fraction be 0.7%-0.83% triethylamine, gradient washes
It is de-；
The gradient elution specifically:
0-3min, mobile phase A are increased to 16% by volume fraction for 11%, and Mobile phase B is reduced to by volume fraction for 89%
84%；
3min-9min, keeping mobile phase A volume fraction is 16%, and keeping Mobile phase B volume fraction is 84%；
9min-20min, mobile phase A are increased to 20% by volume fraction for 16%, and Mobile phase B is 84% reduction by volume fraction
To 80%；
20min-34min, keeping mobile phase A volume fraction is 20%, and keeping Mobile phase B volume fraction is 80%；
34min-40min, mobile phase A are increased to 80% by volume fraction for 20%, and Mobile phase B is 80% reduction by volume fraction
To 20%；
40min-41min, mobile phase A are reduced to 11% by volume fraction for 80%, and Mobile phase B is 20% raising by volume fraction
To 89%；
41min-50min, it is 11% that mobile phase A, which keeps volume fraction, and it is 89% that Mobile phase B, which keeps volume fraction,.
3. construction method according to claim 2, which is characterized in that the chromatographic condition of the ultra performance liquid chromatography also wraps
It includes:
28 DEG C -32 DEG C of column temperature, flow velocity is 0.28mL-0.32mL per minute, Detection wavelength 254nm-360nm.
4. construction method according to claim 1, which is characterized in that the Extraction solvent is that volume fraction is 60%-
80% methanol aqueous solution, dosage are that 50mL-100mL is added in every 1g eclipta medicinal material.
5. construction method according to claim 1-4, which is characterized in that the time being heated to reflux is
60min-90min。
6. a kind of detection method of eclipta medicinal material, which comprises the following steps:
The preparation of reference solution: respectively with wedelolactone and 4, bis--O- caffeoylquinic acids of 5- are reference substance, and solubilizer is molten
Solution, prepares reference substance reference solution；Extraction solvent is added into eclipta control medicinal material, is heated to reflux, obtains extracting solution, by institute
Extracting solution filtration is stated, takes subsequent filtrate as control medicinal material reference solution；
The preparation of testing sample solution: Extraction solvent being added into sample to be tested, is heated to reflux, and obtains extracting solution III, mentions described
It takes liquid III to filter, takes subsequent filtrate as testing sample solution；
Measurement: the reference substance reference solution, control medicinal material reference solution, testing sample solution are injected into ultra high efficiency liquid phase
It is measured in chromatograph.
7. detection method according to claim 6, which is characterized in that the chromatographic condition packet of the ultra performance liquid chromatography
It includes:
Stationary phase: using octadecylsilane chemically bonded silica as the chromatographic column of filler；
Mobile phase: mobile phase A is acetonitrile, and Mobile phase B is triethylamine phosphate buffer, and the triethylamine phosphate buffer is with water
Solvent, comprising volume fraction be 0.5%-0.6% phosphoric acid and volume fraction be 0.7%-0.83% triethylamine, gradient washes
It is de-；
The gradient elution specifically:
0-3min, mobile phase A are increased to 16% by volume fraction for 11%, and Mobile phase B is reduced to by volume fraction for 89%
84%；
3min-9min, keeping mobile phase A volume fraction is 16%, and keeping Mobile phase B volume fraction is 84%；
9min-20min, mobile phase A are increased to 20% by volume fraction for 16%, and Mobile phase B is 84% reduction by volume fraction
To 80%；
20min-34min, keeping mobile phase A volume fraction is 20%, and keeping Mobile phase B volume fraction is 80%；
34min-40min, mobile phase A are increased to 80% by volume fraction for 20%, and Mobile phase B is 80% reduction by volume fraction
To 20%；
40min-41min, mobile phase A are reduced to 11% by volume fraction for 80%, and Mobile phase B is 20% raising by volume fraction
To 89%；
41min-50min, it is 11% that mobile phase A, which keeps volume fraction, and it is 89% that Mobile phase B, which keeps volume fraction,.
8. detection method according to claim 7, which is characterized in that the chromatographic condition of the ultra performance liquid chromatography also wraps
It includes:
28 DEG C -32 DEG C of column temperature, flow velocity is 0.28mL-0.32mL per minute, Detection wavelength 254nm-360nm.
9. detection method according to claim 6, which is characterized in that the Extraction solvent is that volume fraction is 60%-
80% methanol aqueous solution, dosage are that 50mL-100mL is added in every 1g eclipta medicinal material.
10. according to the described in any item detection methods of claim 6-9, which is characterized in that the time being heated to reflux is
60min-90min。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113917004A (en) * | 2021-08-31 | 2022-01-11 | 湖北瑞华制药有限责任公司 | Quality detection method for polygala tenuifolia medicinal material |
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