CN107389821A - A kind of method of active ingredient in measure ageratum oral liquid - Google Patents
A kind of method of active ingredient in measure ageratum oral liquid Download PDFInfo
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- CN107389821A CN107389821A CN201710613288.3A CN201710613288A CN107389821A CN 107389821 A CN107389821 A CN 107389821A CN 201710613288 A CN201710613288 A CN 201710613288A CN 107389821 A CN107389821 A CN 107389821A
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- magnolol
- honokiol
- aurantiamarin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The present invention provides one kind using two benches linear gradient elution method to the method for measuring simultaneously of three kinds of aurantiamarin, magnolol and honokiol component contents in ageratum oral liquid.The method of the present invention is applicable high performance liquid chromatography（HPLC）, Ultra Performance Liquid Chromatography instrument（UPLC）And other form and aspect chromatographs, and suitable for the octadecylsilane chemically bonded silica of various models and specification（C18）For the chromatographic column of filler.The inventive method is equally applicable to three kinds of aurantiamarin in HuoXiangZhengQiShui, magnolol and honokiol component contents while determined, i.e., the inventive method is applied to the quality control of ageratum oral liquid and HuoXiangZhengQiShui.The inventive method versatility in the chromatographic column that various liquid chromatographs and C18 are filler is good, and sample preparation methods are simple, stability is high, easy to operate, safely, fast and accurately reliable, efficiency high.
Description
Technical field
The present invention relates to component content method for measuring in ageratum oral liquid, and in particular to a kind of ageratum is oral
Active ingredient method for measuring simultaneously in liquid, belong to the detection method field of Chinese patent drug composition.
Background technology
Come from the Song Dynasty in ageratum oral liquid original side《The formulary of peaceful benevolent dispensary》, its active ingredient be magnolol and
Magnolol and aurantiamarin.Developed by the plus-minuss of thousand, transforming one kind as by modern crafts is easy to carry and the eutherapeutic modern times
Type preparation.We relieving summer-heat of removing dampness by means of aromatics, relieving exterior syndrome and dispelling cold, keep away dirty preventing or arresting vomiting using Pogostemon cablin as monarch, please spleen with, and Zhi Biaoli；It is purple
Soviet Union, radix angelicae is used as the minister materials, fragrance walks to alter, and pungent scattered temperature is logical, relieving exterior syndrome and dispelling cold and and dampness elimination heresy.Dried orange peel qi-regulating eliminating dampness with, be good for by Poria cocos, rhizoma atractylodis
Spleen dampness removing, shell of areca nut promoting the circulation of qi dampness removing, balloonflower root facilitaing lung profit diaphragm, ginger, regulating spleen and stomach are adjutant altogether, and radix glycyrrhizae coordinating the drug actions of a prescription is to make.Tool
Have inducing diaphoresis dampness dissipating, QI regulating and it is middle the effect of, for catching cold, internal injury humidity hysteresis or summer hinder flu caused by summer-heat and damp, symptoms include headache
Dusk is heavy, chest diaphragm spleen is vexed, abdominal distention, vomiting and diarrhea, or common cold of gastrointestinal type symptom.
Version in 2015《Pharmacopoeia of People's Republic of China》Ageratum oral liquid, HuoXiangZhengQiShui, the ageratum included
Soft capsule and ageratum dripping pill, using high performance liquid chromatography to the aurantiamarin in dried orange peel, the honokiol in the bark of official magnolia and
Three kinds of compositions of magnolol carry out assay.Wherein three kinds of ageratum oral liquid, HuoXiangZhengQiShui, HuoXiangZhengQi soft capsule productions
Product carry out assay using the method for fractional steps to aurantiamarin, honokiol, magnolol, and ageratum dripping pill uses linear gradient elution method
Assay disposably is carried out to aurantiamarin, honokiol, magnolol, but its gradient elution use time control, instrument difference,
Chromatographic column is different, after chromatographic condition change, as a result can not reappear.Ageratum series of products assay is required to detect orange peel
Three kinds of glycosides, honokiol, magnolol compositions, chromatographic column filler is octadecylsilane chemically bonded silica, but mobile phase is different,
Test sample prepares more complicated, has used toxic reagent chloroform again；And detection time length be present, the problems such as efficiency is low.It is right
The elution program that Chinese medicine multicomponent determines simultaneously, using stationary chromatographic post type and specification, fix elution time and adjust flowing
Phase Proportion carries out analysis detection, it is maximum the defects of be versatility of the method in different type, the chromatographic column of different size and again
Existing property extreme difference, method can not use after even more changing chromatographic column.Also have document report using high performance liquid chromatography to wrinkled giant hyssop just
3,4,5 in gas series of products, or even 7 compositions are detected, belong to pilot study, method poor universality,
Do not have yet in effective workaround actual application it is simple, easily and fast, the problem of versatility is good.
Ageratum series of products market demand and facility capacity are all very big, how to establish simple, quick, versatility
Good detection method, it is major issue of the pendulum in face of researcher.The present invention is based on produce reality problem, to ageratum
The assay of series of products is studied, establish test sample preparation method, 3 kinds of index/active ingredient (aurantiamarin and
Magnolol, magnolol) content assaying method.The inventive method with《Chinese Pharmacopoeia》Method mark glue, the letter of test sample preparation method
It is single, avoid the use of toxic reagent chloroform；Detection method is simple, quick, accurate, stably, to various instruments and chromatogram
Post versatility is good, drastically increases efficiency.This method can apply to ageratum oral liquid, HuoXiangZhengQiShui, ageratum
The assay of soft capsule and ageratum dripping pill.
The content of the invention
For deficiencies of the prior art, it is an object of the invention to provide one kind to determine ageratum oral liquid
The method of middle active ingredient, solve test sample and prepare more complicated and the unsafe problem of toxic reagent, and existing inspection be present
Survey method has that detection time is long, mobile phase differs, and complex operation, efficiency are low, versatility and the problem of reappearance extreme difference.
Above-mentioned purpose is realized, the present invention adopts the following technical scheme that：Active ingredient in one kind measure ageratum oral liquid
Method, it is characterised in that comprise the following steps：
1) preparation of standard reference material solution：
Take aurantiamarin, magnolol and honokiol standard reference material appropriate, it is accurately weighed, add methanol to be configured to every 1mL and contain
Aurantiamarin, magnolol and honokiol are respectively 0.05~0.30mg mixed solution, and 0.2~0.5 μm of membrane filtration is to be measured；
2) preparation of need testing solution：
Take ageratum oral liquid stoste or stoste is diluted 2-5 times with 60~100% methanol/ethanol；0.2~0.5 μ
M membrane filtrations, it is to be measured；
3) determined using high performance liquid chromatograph：
Step 1) is put into high performance liquid chromatograph with the standard liquid and need testing solution 2) prepared, using two benches
Linear gradient elution method determines, and chromatographic condition is as follows：
Chromatographic column is C18 chromatographic columns, and column temperature is 25~40 DEG C, and mobile phase is methanol-acetonitrile-water, and flow velocity is 0.2~2mL/
Min, sample size are 2~20 μ L (determining actual sample size, flow velocity according to liquid chromatograph and chromatographic column specification)；Gradient collapse journey
Sequence is carried out according to the following table：
4) three kinds of aurantiamarin, magnolol and honokiol cubages in test sample：
It is control first with the HPLC chromatogram peak obtained by aurantiamarin, magnolol and honokiol standard reference material solution,
Standard control collection of illustrative plates is prepared, and establishes the linear equation of concentration and integral area；Then with external standard method with standard liquid chromatogram
For control, aurantiamarin, the content of three kinds of compositions of magnolol and honokiol in test sample are calculated.
Wherein, two benches linear gradient elution method is in the step 3)：Aurantiamarin contains in first stage detection testing sample
Amount, after aurantiamarin chromatographic peak appearance terminates, mobile phase ratio is adjusted, carries out second stage analysis；Second stage detection is to be measured
Two kinds of component contents of magnolol and honokiol, after the chromatographic peak appearance of magnolol and honokiol terminates, i.e., complete in sample
Into a sample analysis；Then after chromatogram column equilibration to first stage mobile phase, next sample analysis can be carried out.
The mobile phase reclaimed water contains the phosphoric acid contained in formic acid or acetic acid, or water less than 0.7% less than 0.1%.
Liquid chromatograph described in the step 3) is high performance liquid chromatograph (HPLC), Ultra Performance Liquid Chromatography instrument
(UPLC), medium pressure liguid chromatograph or low pressure liquid phase chromatography instrument.
The present disclosure additionally applies for for aurantiamarin, honokiol, Determination of Magnolol measure in HuoXiangZhengQiShui, i.e., method is fitted
Assay for HuoXiangZhengQiShui active ingredient.
Compared with prior art, the present invention has the advantages that：
1st, it is dilute using test sample stoste or test sample stoste methanol or ethanol this invention simplifies sample-pretreating method
Filtered after releasing certain multiple, be directly used in efficient liquid phase chromatographic analysis, test sample prepared by this method within 120 hours still
With stability, no longer using chloroform extraction, harm of the use of toxic reagent to human body and environment is avoided, also significantly
Manpower and time cost are saved；Sample pre-treatments step is reduced simultaneously, reduces human factor shadow to caused by analytical structure
Ring, therefore the degree of accuracy of detection can be improved.
2nd, the present invention passes through the processing method to test sample and the screening of chromatographic condition so that the inventive method has good
Good specificity, accuracy, reappearance and accuracy, reduces the occupancy of instrument resource, greatlys save manpower and time
Cost, hence it is evident that improve detection efficiency.
3rd, the present invention is analyzed sample using two benches gradient elution program, and the first stage detects a kind of composition (orange peel
Glycosides), second stage detects 2 kinds of compositions (honokiol and magnolol), i.e. the survey of Multiple components content is completed in a chromatography
It is fixed, and this method is applied to different type and the chromatographic column of different size, selects flash chromatography post to improve test sample analysis
Efficiency, and can reach preferable analytical effect.Therefore the present invention greatly shortens measure analysis time, the 20min on HPLC
Within complete the measure of 3 kinds of component contents of a sample, shorter, the tested 3 kinds of composition chromatograms of detection time are analyzed on UPLC
The RSD that peak has good separating degree, precision of method, repeatability is respectively less than 2.0%.It is unfixed in elution process to be washed per the stage
The de- time.Therefore, method of the invention is applied in the chromatographic column of different liquid chromatographs, different type and different size, table
Reveal good versatility.Quality evaluation and control available for ageratum oral liquid and ageratum aquatic products；Also can use
In the quality analysis and control of HuoXiangZhengQi soft capsule and ageratum dripping pill, because HuoXiangZhengQi soft capsule and ageratum drip
Ball is not liquid, so sample-pretreating method is different, but sample analysis method is the same.
4th, the present invention is set several isocratic or terraced using several chromatographic peaks with indicative (or index) composition as reference
Eluant, eluent ratio is spent, forms complete gradient elution program, carries out multicomponent content measure or fingerprint map construction simultaneously, each
The elution time in stage determines according to instrument, flow velocity, chromatographic column type and specification, therefore, in different instruments, different type and not
Good versatility in the chromatographic column of same specification.This is the multicomponent of plant (including Chinese medicine) and Chinese medicine compound prescription while contains measurement
Fixed or structure fingerprint map construction has established theoretical foundation.
Brief description of the drawings
Fig. 1 is system suitability test Plays product Solution H PLC figures；
Fig. 2 is that test sample product Solution H PLC schemes in system suitability test；
Fig. 3 is that the negative sample Solution H PLC without aurantiamarin schemes in specificity experiment；
Fig. 4 is the negative sample Solution H PLC figures without honokiol and magnolol in specificity experiment；
Fig. 5 is that need testing solution HPLC schemes in specificity experiment；
Fig. 6-15 is the HPLC figures of method 1-10 analysis need testing solutions respectively；
Figure 16-22 is the HPLC figures of different chromatographic column A-G analyses need testing solutions respectively；
Figure 23 is 20 DEG C of analysis need testing solution HPLC figures；
Figure 24 is 25 DEG C of analysis need testing solution HPLC figures；
Figure 25 is 30 DEG C of analysis need testing solution HPLC figures；
Figure 26 is 35 DEG C of analysis need testing solution HPLC figures；
Figure 27 is 40 DEG C of analysis need testing solution HPLC figures.
Embodiment
The present invention is described in further detail with reference to specific embodiments and the drawings.
Instrument and reagent
Highly effective liquid phase chromatographic system：The high performance liquid chromatographs of Agilent 1260 (prepare vacuum degassing machine and quaternary pump
G1311C, column oven G1316A, automatic sampler G1329B, PDAD G4212B).Excellent general serial ultra-pure water
Device：Sichuan UP Hyperpure Technology Co., Ltd..Supersonic wave cleaning machine：NingBo XinZhi Biology Science Co., Ltd.
Reference substance is purchased from Chinese food drug assay research institute, (1) aurantiamarin lot number：110721-201316；(2) and
Magnolol lot number：110730-201313；(3) magnolol lot number：110729-200412.Ageratum oral liquid test sample lot number
For：16111406th, 16090943~16090946,16090949~16090951,16091025~16091027,16091030
~16091033, provided by Taiji Group Fuling Pharmaceutical Factory Co., Ltd.HuoXiangZhengQiShui is 6 different factories in pharmacopeia purchase
Family's product is as test sample.Methanol and acetonitrile are chromatographically pure, and water is secondary deionized water, and remaining reagent is that analysis is pure.
Embodiment 1
The method of active ingredient (while determines aurantiamarin/magnolol/honokiol in a kind of measure ageratum oral liquid
The method of content)
The preparation of standard reference material solution：Aurantiamarin, magnolol and honokiol standard reference material accurately are weighed, is configured to
Mixed reference substance solution shown in table 1,0.45 μm of membrane filtration are standby.
The reference substance solution of table 1
The preparation of need testing solution：Accurately measure ageratum oral liquid stoste 5.0mL to insert in 10mL volumetric flasks, use first
Alcohol is settled to scale, and 0.45 μm of membrane filtration, standby after mixing.
(1) system suitability test
Reference substance solution and need testing solution are taken, chromatographic condition is：(1) chromatographic column：Water generation Xbridge C18,3.5 μ
M, 4.6 × 100mm；(2) column temperature：35℃；(3) sample size：5μL；(4) flow velocity：1.00mL/min；(5) mobile phase：Acetonitrile-first
The aqueous formic acid of alcohol -0.4%, gradient elution program are as shown in table 2；(6) Detection wavelength：Aurantiamarin Detection wavelength is 283nm, and
The Detection wavelength of magnolol and magnolol is 294nm.
The gradient elution program of table 2
As a result as depicted in figs. 1 and 2,3 kinds of compositions to be measured can reach baseline separation, big with the separating degree of adjacent chromatographic peak
In 5.0, the theoretical number of plates is calculated more than 8000 with aurantiamarin chromatographic peak, theoretical tower is calculated with honokiol and magnolol chromatographic peak
Plate number is more than 70000, and peak type is good.
Number of theoretical plate is calculated by aurantiamarin chromatographic peak should be not less than 5000, and number of theoretical plate presses magnolol or honokiol chromatogram
Peak, which calculates, should be not less than 20000.
(2) linear relationship is investigated
Accurate weighed aurantiamarin (lot number：110721-201316, in terms of 95.3%), honokiol (lot number：110730-
201313, in terms of 99.1%) and magnolol (lot number：110729-200412, in terms of 99.0%) each 10mg of reference substance, accurately matches somebody with somebody
Be made 1.00,0.60,0.40,0.30,0.20,0.10,0.06,0.04,0.03,0.02,0.01mg/mL solution.According to system
Chromatographic condition under employment and suitability test (E ＆ ST) item is measured, and concentration and integral area (Y) are established to sample size with integral area (Y)
Regression equation (table 3).
The equation of linear regression of table 3
From table 3 it can be seen that signal to noise ratio when detecting minimum sample concentration under this methodology is more than 10, aurantiamarin and thickness
The concentration of plain phenol honokiol is linear good in the range of 0.010~1.00mg/mL.《Chinese Pharmacopoeia》Provide that ageratum is oral
Content of hesperidin must not be less than 0.1mg/mL in liquid, and honokiol honokiol accumulated dose must not be less than 0.3mg/mL, therefore should
The range of linearity meets the assay requirement of ageratum oral liquid.
(3) specificity is tested
This product test sample, the negative sample without aurantiamarin and the negative sample without honokiol and magnolol are taken, is pressed
Sample treatment processing is drafted, is prepared into need testing solution, negative sample solution.According to the color under system suitability item
Spectral condition, HPLC detections are carried out to negative controls and positive test sample.
As a result as in Figure 3-5, the negative sample without aurantiamarin and the negative sample without honokiol and magnolol
Noiseless at main peak, method specificity is good.
(4) precision test
Standard reference material solution and 3 parts of test sample solution are taken, according to the chromatographic condition under system suitability item, is connected
Continuous sample introduction, different personnel, the not precision of same date and different Instrumental results are calculated according to measurement result respectively.
3 different personnel are measured to aurantiamarin, honokiol and Determination of Magnolol in test sample, and RSD is respectively
0.68%th, 1.07% and 0.99%, show that different personnel are good using this method measurement result precision；For three days on end to for trying
Aurantiamarin, honokiol and Determination of Magnolol measure, RSD values are respectively 0.79%, 1.14% and 0.99% in product, are shown in the daytime
Precision is good；3 different instruments are measured to aurantiamarin, honokiol and Determination of Magnolol in test sample, RSD values difference
For 0.75%, 0.52% and 0.85%, show that different Instrumental results precision are good.
(5) replica test
According to the preparation method of test sample, sample (lot number is taken：16111406) 7 parts of need testing solutions are prepared, according to system
Chromatographic condition under employment and suitability test (E ＆ ST) item, the amount of each composition is calculated according to standard reference material.
As a result the RSD of aurantiamarin, honokiol and magnolol is respectively 0.62%, 0.95% and 0.75% in test sample,
Show that method repeatability is good.
(5) stability test
According to the preparation method of test sample, sample (lot number is taken：16111406) 3 parts of need testing solutions are prepared, according to system
Chromatographic condition under employment and suitability test (E ＆ ST) item is analyzed.Wherein test sample solution closed preservation at room temperature, deposit 120h.
The RSD of aurantiamarin, honokiol and magnolol retention time is respectively 1.60%, 0.76% and 1.10%, chromatogram
The RSD of peak area is respectively 0.34%, 0.50% and 0.21%, shows that the need testing solution of preparation is good in 120h internal stabilities
It is good.
(7) recovery test
Accurate weighed aurantiamarin (lot number respectively：110721-201316, in terms of 95.3%) 12.0mg, honokiol (batch
Number：110730-201313, in terms of 99.1%) 11.0mg, magnolol (lot number：110729-200412, in terms of 99.0%)
10.0mg, aurantiamarin 0.24mg/mL, honokiol 0.22mg/mL and magnolol 0.20mg/ are configured to after being dissolved with methanol respectively
ML standard liquid.Take 10mL volumetric flasks, accurately add successively into need testing solution respectively 3 compositions to be measured (aurantiamarin and
Magnolol and magnolol) amount about 0.5,1.0 and 1.5 times of amount standard substance, again with methanol is settled to scale, is applicable according to system
Property experiment item under chromatographic condition analyzed.
As a result aurantiamarin, honokiol, the average recovery rate of magnolol are respectively 99.55%, 99.19% and 99.41%,
RSD is respectively 0.56%, 0.74% and 1.00%.
Embodiment 2
Parameter optimization
(1) test sample preparation method
Test sample stoste with 1 times, 2 times, 2.5 times, 5 times of methanol or methanol dilution and is not diluted, obtains 5 kinds of test samples,
Analyzed according to the chromatographic condition under system suitability item, as a result the content of aurantiamarin, honokiol and magnolol is equal
No significant difference, illustrate that solvent and extension rate will not impact to testing result.Respectively with water, 20%, 40%, 60%,
80% and 100% methanol and ethanol dilutes test sample twice, is carried out according to the chromatographic condition under system suitability item
Analysis, as a result when the concentration of methanol and ethanol is less than 60%, with test sample stoste and 100% methanol or ethanol dilution metering knot
Fruit contrasts, and the content of measure aurantiamarin, honokiol and magnolol is less than normal.Therefore, it is dilute with more than 80% methanol or ethanol
Releasing test sample is advisable.
(2) mobile phase is investigated
Methanol-water, acetonitrile-water, and methanol-acetonitrile-water difference flow phase system have been investigated respectively, as a result methanol-water
It is not so good as methanol-acetonitrile-water to the separation situation of 3 kinds of compositions to be measured in test sample with acetonitrile-water, therefore selects methanol-acetonitrile-water
System；The influence of formic acid, acetic acid and phosphoric acid to composition chromatographic peak to be measured is investigated, as a result the appropriate content of these three acid is to orange peel
Glycosides, honokiol, magnolol have preferable separating effect.When wherein pH value of water solution is less than 2.7, good separating effect.But pH
Being worth too low can influence chromatogram column life.Therefore the formic acid solution or 0.02~0.10% phosphoric acid of this method preferably 0.3~0.6%
Solution.
(3) selection of gradient condition
Using the aqueous formic acid of methanol-acetonitrile -0.4% as mobile phase, set different gradient elution program method for building up 1~
10, specifically it is shown in Table shown in 4~13.Other chromatographic conditions：(1) chromatographic column：Water generation Xbridge C18, 3.5 μm, 4.6 ×
100mm；(2) column temperature：35℃；(3) sample size：5μL；(4) flow velocity：1.00mL/min；(5) Detection wavelength：Aurantiamarin detects ripple
The Detection wavelength of a length of 283nm, honokiol and magnolol is 294nm.
The method 1 of table 4
The method 2 of table 5
The method 3 of table 6
The method 4 of table 7
The method 5 of table 8
The method 6 of table 9
The method 7 of table 10
The method 8 of table 11
The method 9 of table 12
The method 10 of table 13
When wherein each gradient elution program method of application carries out HPLC analyses, all formed by two isocratic flowings are combined
Two analysis phases, the first stage is the detection of aurantiamarin in test sample, after aurantiamarin chromatographic peak appearance terminates, mobile phase
Second stage analysis is carried out after adjustment；Second stage is the detection of honokiol and magnolol in test sample, treats honokiol
And after magnolol chromatographic peak appearance terminates, complete analysis；Then with after first stage mobile phase ratio balance chromatographic column, can carry out
Next sample analysis.Each gradient elution program method can be to aurantiamarin, honokiol and thickness in ageratum oral liquid
Plain phenol carries out assay.Each stage specific elution time is determined according to instrument, flow velocity, column temperature, chromatographic column specification etc..Root
It is the chromatographic condition under the options according to gradient condition according to method 1~10, after analyzing test sample chromatogram, is determined after optimization
Elution time.
Compare the chromatographic peak of 3 compositions to be measured of distinct methods measure, and record per each and every one chromatographic peak collection of illustrative plates (Fig. 6~15)
Relevant parameter is shown in Table 14.
Chromatographic performance parameter of the distinct methods of table 14 to three kinds of composition detections to be measured
According to chromatogram peak symmetry, separating degree and the theoretical cam curve of 3 kinds of compositions to be measured in chromatogram 6~15 and table 14 point
Data are analysed, judge separating property of the different gradient elution programs to this 3 kinds of compositions.As a result show, various methods are to be measured to three kinds
Composition can carry out analysis detection.But in methanol-water and acetonitrile-water system, the separation of composition chromatographic peak and adjacent peak to be measured
Spend it is poor, and impurity peaks interference it is more obvious；And three kinds of compositions to be measured have good separation in methanol-acetonitrile-water system
Effect.
According to above-mentioned analysis result, determine in the aqueous formic acid system of methanol-acetonitrile -0.4%, methanol, acetonitrile and water exist
Each solvent ratios in gradient elution program are shown in Table 15.The wherein improved generalized gradient elution program such as table of method for optimizing 1
Shown in 16.
The gradient elution program of table 15 and Detection wavelength
The preferred gradient elution program of table 16 and Detection wavelength
(4) different chromatographic columns compare
Select different manufacturers, different model, 7 root chromatogram column A~F of different size：(A)Agilent Eclipse
Pluse c18 chromatographic columns (3.5 μm, 4.6mm × 100mm)；(B) Waters symmetry C18 chromatographic columns (3.5 μm, 4.6mm
×75mm)；(C) Agilent Zorbax SB-C18 chromatographic columns (5 μm, 4.6mm × 150mm)；(D)Agilent Eclipse
XDB C18 chromatographic columns (5 μm, 4.6mm × 250mm)；(E) YMC-Pack ODS-A chromatographic columns (5 μm, 4.6mm × 250mm)；F)
Shim-Pack VP-ODS chromatographic columns (5 μm, 4.6mm × 250mm)；(G) Waters XBridge C18 chromatographic columns (3.5 μm,
4.6mm×100mm).Other chromatographic conditions：Column temperature is 35 DEG C, and sample size is 5 μ L or 10 μ L (being determined according to chromatographic column specification),
Flow velocity is 1.00mL/min；Gradient elution program method as shown in table 16.Test sample solution is analyzed with HPLC, obtains color
Spectrogram (Figure 16~22), it is as shown in table 17 further to obtain aurantiamarin, honokiol, magnolol chromatographic determination performance parameter.
Chromatographic column A~the F of table 17 detects the chromatographic data of three kinds of compositions to be measured
As can be seen from Table 17, under identical chromatographic conditions, 7 different chromatographic columns are treated to 3 in test sample more than
Survey composition and carry out HPLC detection and analysis, show good separating effect, peak shape piles that symmetrical, baseline is steady, illustrates the party
Method has good applicability, and the theoretical cam curve of composition aurantiamarin honokiol chromatographic peak wherein to be measured to different chromatographic columns
It is superior to《Chinese Pharmacopoeia (2015)》The numerical value 2000 and 5000 of defined.Chromatographic column filler particle diameter is smaller, chromatographic column post effect
It is higher；It is faster compared with small particle size filler and shorter chromatographic column, analyze speed.Chromatographic column filler particle diameter is 3.5 μm, column length≤
100mm, it is to belong to flash chromatography post.Selection flash chromatography post can improve testing sample analysis efficiency, and and can, which reaches, preferably divides
Analyse effect.Therefore on the premise of analysis efficiency and effect is taken into account, preferred filler particle diameter is smaller, column length is shorter flash chromatography
Post.According to result of the test, although flash chromatography post packing material size is small, column length is short, and chromatogram column pressure is not high, suitable for current
Conventional high performance liquid chromatograph.
(5) selection of column temperature
According to the chromatographic condition under system suitability item, preferred gradient elution program (as shown in table 16) is selected,
Column temperature is respectively at 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C, and HPLC analyses, obtained chromatogram are carried out to same need testing solution
Scheme (Figure 23~27), it is as shown in table 18 further to obtain aurantiamarin, honokiol and magnolol chromatographic determination performance parameter.
The chromatographic data of three kinds of compositions to be measured is detected under the conditions of 18 different column temperatures of table
As can be seen from Table 18, investigating in temperature range, with the rise of column temperature, chromatographic peak retention time shifts to an earlier date, when
When temperature is raised to more than 35 DEG C, chromatographic peak retention time shifts to an earlier date unobvious；With the rise of column temperature, under chromatographic column post pressure is obvious
Drop；The separating degree of chromatographic peak and adjacent peak with temperature rise slightly raise after again on a declining curve, the symmetry and reason of chromatographic peak
Obvious reduce does not occur by the number of plates.To sum up analyze, preferably select column temperature as 30~35 DEG C.
Embodiment 3
The sample of 21 different batches (is provided, 21) lot number is shown in Table, adopted by Taiji Group Fuling Pharmaceutical Factory Co., Ltd
With the inventive method and《Chinese Pharmacopoeia (2015)》Method determines aurantiamarin, honokiol in ageratum oral liquid respectively
And Determination of Magnolol is measured respectively.The two methods of data judging obtained according to analysis whether there is significant difference.
Follow the steps below analysis.
Standard solution is prepared：Accurate weighed aurantiamarin, honokiol and magnolol standard reference material, use methanol dilution
Afterwards, the mixed reference substance solution of 2 parts of various concentrations is prepared, as shown in table 19：
The reference substance solution of table 19
1st, analyze using the inventive method and (be labeled as method A)
1) prepared by need testing solution：Accurate measuring test sample stoste 5mL is transferred in 10mL measuring bottles, with methanol constant volume to quarter
Degree, shakes up, 0.22 μm of membrane filtration, takes subsequent filtrate, to be measured.
2) high performance liquid chromatograph bioassay standard reference substance solution and test sample solution are used：By standard reference material solution and
Test sample solution injects high performance liquid chromatograph, obtains chromatogram.
Chromatographic condition during efficient liquid phase chromatographic analysis is：(1) chromatographic column：Water generation Xbridge C18,3.5 μm,
4.6×100mm；(2) column temperature：35℃；(3) sample size：5μL；(4) flow velocity：1.00mL/min；(5) mobile phase：Acetonitrile-methanol-
0.4% aqueous formic acid, gradient elution program are as shown in table 20.
The gradient elution program of table 20
3) according to chromatogram, the content of aurantiamarin, honokiol and magnolol in test sample solution is calculated with external standard method,
And according to the content of aurantiamarin, honokiol and magnolol in extension rate conversion test sample.
2nd, use《Chinese Pharmacopoeia (2015)》Method analyzes (being labeled as method B)
1) prepared by need testing solution：When determining Determination of hesperidin in pericarpium citri reticulatae, accurate measuring test sample stoste 10mL is transferred to
In 25mL measuring bottles, with methanol constant volume to scale, shake up, filter, produce；When honokiol is with Determination of Magnolol in the measure bark of official magnolia,
Accurate measuring test sample stoste 5mL, adds hydrochloric acid 2 to drip, and with 10mL chloroform extractions 3 times, merges chloroform liquid, is evaporated, residual
Slag is dissolved with methanol, is transferred in 10mL measuring bottles, with methanol constant volume to scale, is shaken up, and filtration, is produced；
2) high performance liquid chromatograph bioassay standard reference substance solution and need testing solution are used：By standard reference material solution and confession
Test sample solution injects high performance liquid chromatograph, obtains chromatogram.
Chromatographic condition during efficient liquid phase chromatographic analysis is：(1) chromatographic column：Agilent Eclipse XDB C18
Chromatographic column (5 μm, 4.6mm × 250mm)；(2) column temperature：35℃；(3) flow velocity：1.00mL/min；(4) mobile phase and Detection wavelength：
The mobile phase of aurantiamarin detection is the acetum of methanol -0.4%, Detection wavelength 283nm；What honokiol and magnolol detected
Mobile phase is methanol-isopropanol-water (36:21:36), Detection wavelength 294nm；(5) sample size：Sample introduction when aurantiamarin detects
Amount, it is accurate respectively to draw reference substance and each 5 μ L of need testing solution, inject liquid chromatograph；When honokiol and magnolol detect
Sample size, it is accurate respectively to draw reference substance and each 10 μ L of need testing solution, inject liquid chromatograph.
3) according to chromatogram, the content of aurantiamarin, honokiol and magnolol in need testing solution is calculated with external standard method, and
According to the content of aurantiamarin, honokiol and magnolol in extension rate conversion test sample.
Method A and method B in accordance with the above, to 21 batches (16090939~16090952 and 16091025~
16091033) aurantiamarin, honokiol and the magnolol in ageratum oral liquid product has carried out assay, as a result such as
Shown in table 21.Method A the data obtaineds are completed by this laboratory, and method B the data obtaineds are by Taiji Group Fuling Pharmaceutical Factory matter
Inspection center provides.When being determined to content of hesperidin in test sample, prepared by method A and method B test sample solution, be to supply
Test product stoste uses 2 times and 2.5 times of methanol dilution respectively, and in theory, test sample extension rate will not cause shadow to testing result
Ring, so both approaches acquired results should no significant difference.
HPLC assay results of the method A of table 21 and method B to 3 kinds of compositions in 21 batch ageratum oral liquids
As can be seen from Table 21, measurement result shows in 21 batch samples, two methods measure content of hesperidin, its
In the relative deviations of 17 batches be less than 2.0%, the relative deviation of remaining 4 batch is less than 3.0%, shows obtained by two methods
Testing result difference it is small, can meet testing requirements (in ageratum oral liquid containing dried orange peel in terms of aurantiamarin, must not be less than
0.10mg/mL).The difference of above two method measurement result is mainly derived from human error caused by human users' difference,
It is likely to be instrument, systematic error existing for method.
When honokiol in sample and Determination of Magnolol determine, it is obvious that both approaches measure result difference.Method A
Ratio method B measures honokiol and is higher by 3.67%~17.45% with magnolol total content, is averagely higher by 9.04%.Because for examination
The difference of sample preparation methods can be produced to testing result and significantly affected, and honokiol and magnolol in this product are detected with method B
During content, sample chloroform extraction, it may be possible to caused by chloroform extraction is incomplete；And method A be directly will be to be measured
Product, as a result should be more accurate with being detected after methanol dilution.Two methods can meet testing requirements (containing thickness in ageratum oral liquid
Piao must not be less than 0.30mg/mL in terms of the total amount of Magnolol and Honokiol).
Embodiment 4
Using method A in embodiment 3 to HuoXiangZhengQiShui carry out quality analysis, detection wherein aurantiamarin, honokiol and
The content of magnolol.Test sample preparation method is：The HuoXiangZhengQiShui stoste of accurate measuring 5mL different manufacturers production, is transferred to
It is to be measured with methanol constant volume to scale, 0.2 μm of membrane filtration in 10mL volumetric flasks.Side in detection method and step such as embodiment 3
Described in method A, testing result is as shown in table 22.
The HPLC measurement results of 3 component contents in 226 different manufacturers HuoXiangZhengQiShuis of table
According to《Chinese Pharmacopoeia》Content of hesperidin must not be less than 0.18mg/mL in regulation HuoXiangZhengQiShui, honokiol with
Magnolol total amount meter must not be less than 0.58mg/mL.It is equal that this method detects 6 different manufacturers HuoXiangZhengQiShui index component contents
Meet《Chinese Pharmacopoeia》Defined quality requirement.
To sum up, the inventive method can be used for the quality evaluation and control of ageratum oral liquid and ageratum aquatic products.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to the skill of the present invention
Art scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover at this
Among the right of invention.
Claims (4)
- A kind of 1. method for determining active ingredient in ageratum oral liquid, it is characterised in that comprise the following steps：1) preparation of standard reference material solution：Take aurantiamarin, magnolol and honokiol standard reference material appropriate, it is accurately weighed, add methanol to be configured to every 1mL and contain orange peel Glycosides, magnolol and honokiol are respectively 0.05~0.30mg mixed solution；0.2~0.5 μm of membrane filtration, it is to be measured；2) preparation of need testing solution：Take ageratum oral liquid stoste or stoste is diluted 2-5 times with 60~100% methanol/ethanol；0.2~0.5 μm of filter Membrane filtration, it is to be measured；3) determined using high performance liquid chromatograph：Step 1) is put into high performance liquid chromatograph with the standard liquid and need testing solution 2) prepared, using two benches gradient Elution method determines, and chromatographic condition is as follows：Chromatographic column is C18 chromatographic columns, and column temperature is 25~40 DEG C, and mobile phase is methanol-acetonitrile-water, and flow velocity is 0.2~2mL/min, Sample size is 2~20 μ L (determining actual sample size, flow velocity according to liquid chromatograph and chromatographic column specification)；Gradient collapse program is pressed Carried out according to following table：4) three kinds of aurantiamarin, magnolol and honokiol cubages in test sample：It is control first with the HPLC chromatogram peak obtained by aurantiamarin, magnolol and honokiol standard reference material solution, prepares Standard control collection of illustrative plates, and establish the linear equation of concentration and integral area；Then with external standard method using standard liquid chromatogram as pair According to aurantiamarin, the content of three kinds of compositions of magnolol and honokiol in calculating test sample；Wherein, the two benches linear gradient elution method described in step 3) is：The content of aurantiamarin in first stage detection testing sample, After aurantiamarin chromatographic peak appearance terminates, mobile phase ratio is adjusted, carries out second stage analysis；Second stage detects testing sample Middle two kinds of component contents of magnolol and honokiol, after the chromatographic peak appearance of magnolol and honokiol terminates, that is, complete one Individual sample analysis；Then after chromatogram column equilibration to first stage mobile phase, next sample analysis can be carried out.
- 2. the method for active ingredient in ageratum oral liquid is determined according to claim 1, it is characterised in that the flowing Phase reclaimed water contains the phosphoric acid contained in formic acid or acetic acid, or water less than 0.7% less than 0.1%.
- 3. the method for active ingredient in ageratum oral liquid is determined according to claim 1, it is characterised in that the step 3) liquid chromatograph in is high performance liquid chromatograph (HPLC), Ultra Performance Liquid Chromatography instrument (UPLC), medium pressure liguid chromatograph Or low pressure liquid phase chromatography instrument.
- 4. according to the method for active ingredient in any measure ageratum oral liquid of claims 1 to 3, it is characterised in that Aurantiamarin, honokiol, Determination of Magnolol measure suitable for for HuoXiangZhengQiShui, i.e., method has suitable for HuoXiangZhengQiShui Imitate the assay of composition.
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| CN110231418A (en) * | 2019-06-18 | 2019-09-13 | 重庆太极医药研究院有限公司 | A kind of method of Multiple components content in HPLC method separation determination ageratum oral liquid |
| CN114689729A (en) * | 2020-12-31 | 2022-07-01 | 鲁南制药集团股份有限公司 | Method for detecting flavonoid glycoside component in Jingfang granules |
| CN114755338A (en) * | 2022-04-19 | 2022-07-15 | 四川新绿色药业科技发展有限公司 | Detection method of agastache rugosus and preparation thereof |
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| CN116183739A (en) * | 2022-11-25 | 2023-05-30 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Determination method for analyzing fingerprint of Huoxiang Zhengqi soft capsule |
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