CN107677750A - The multicomponent content assaying method of Mussaenda hirsutula - Google Patents
The multicomponent content assaying method of Mussaenda hirsutula Download PDFInfo
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Abstract
The invention discloses a kind of multicomponent content assaying method of Mussaenda hirsutula, is surveyed using one and comments method more（QMSA methods）Determine 6 kinds of organic acid composition neochlorogenic acids, Cryptochlorogenic acid, chlorogenic acids, 3,5 in Mussaenda hirsutula simultaneouslyODCQ, 3,4ODCQ, 4,5ODCQ.The organic acid composition of present invention selection Mussaenda hirsutula carries out assay for index, and precision height, favorable reproducibility, stability is good, the rate of recovery is high, measurement result is accurate, can effectively control the quality of Mussaenda hirsutula.
Description
Technical field
The present invention relates to the multicomponent content assaying method of traditional Chinese medicine detection technique field, specifically Mussaenda hirsutula.
Background technology
Mussaenda hirsutulaMussaenda hirsutuLaMiq. it is climbing shrub, sprig is close soft by rust or grey
Hair.Leaf is to life, and film quality, oval or near avette, long 3~7cm, wide 2~3cm, top is short sharp or tapering, base portion wedge shape, two sides
By sparse pubescence, below and arteries and veins on hair it is closeer.Originate in Hainan, Guangdong, Hunan, Guizhou and Yunnan.It is born at the m of height above sea level 340
In mountain valley, small stream side and wilderness shrubbery, often climb up by holding on in woods on tree crown.This kind of root, leaf can be used as medicine, can heat-clearing, removing toxic substances, antimalarial.Entirely
Strain can heat-clearing, dispelling wind.Mussaenda hirsutula medicinal material is the dry root of Rubiaceae Radix Mussaendae platymiscium Mussaenda hirsutula, stem.
Chemical composition species in Mussaenda hirsutula is similar to Radix Mussaendae, but based on Determination of Organic Acids, cyclenes
Ether terpene methods of glycosides content is relatively low.For Mussaenda hirsutula, in current standard a rationalization differentiate, it is also rarely seen in other patent medicine
Thin layer differentiates, there is not yet the relevant report on its assay；From the point of view of 9 kinds of method for measuring components that Radix Mussaendae is established,
It is simultaneously too high using Determination of Organic Acids and 9 kinds of reference substance costs of iridoid glycoside constituents, it is time-consuming longer, therefore, one need to be established
Content assaying method of the kind for Mussaenda hirsutula.
The content of the invention
Based on this, the invention provides a kind of multicomponent content assaying method of Mussaenda hirsutula, and comment is surveyed using one more
Method, establish one kind while determine 6 kinds of organic acid composition neochlorogenic acids, Cryptochlorogenic acid, chlorogenic acids, 3 in Mussaenda hirsutula,
5-O- DCQ, 3,4-O- DCQ, 4,5-OThe method of-DCQ.
To realize above-mentioned technical purpose, particular content is as follows：
（1）Chromatographic condition：Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as stream
Dynamic phase B, gradient elution is carried out by table 1；The μ L of sample size 10,35 DEG C of column temperature, the mL/min of flow velocity 1.0, the nm of Detection wavelength 328；
The Mussaenda hirsutula gradient elution table of table 1
（2）Extract sample：Take sample powder（Cross No. four sieves）About 0.5 g, it is accurately weighed, put in conical flask with cover, precision adds
The mL of 50% methanol 20, weighed weight, it is ultrasonically treated（The W of power 320, the kHz of frequency 40）30 min, take out, let cool, then it is weighed heavy
Amount, the weight of less loss is supplied with 50% methanol, is shaken up, and is filtered, is taken filtrate, produce；
（3）Prepare neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4-O- DCQs, the coffee acyl Kuis of 3,5-O- bis-
Peaceful acid, 4,5-O- DCQ reference substance storing solutions；
（4）4 kinds of mixed reference substance solutions of different linear concentrations are prepared with above-mentioned 6 kinds of reference substance storing solutions；
（5）It is linear to investigate：Above-mentioned 6 kinds of reference substance storing solutions are pressed（1）Middle μ L of chromatographic condition sample introduction 10,4 kinds of reference substance mixing are molten
Liquid distinguishes the μ L of sample introduction 10, respectively as 5 points of linear concentration from high to low, with the sample size of reference substance（ng）For abscissa,
Peak area is ordinate, draws standard curve；
（6）Precision test：Take with a need testing solution, press（1）Middle chromatographic condition, METHOD FOR CONTINUOUS DETERMINATION 6 times；
（7）Reappearance test：Take with a collection of test sample powder about 0.5g, it is accurately weighed, press（2）Middle extracting method extraction is simultaneously parallel
6 parts of measure；
（8）Accuracy test：Take with a collection of test sample powder about 0.25g, it is accurately weighed, totally 12 parts, calculated by precision result
The content of 9 kinds of compositions out is separately added into three levels（50%、100%、150%）Reference substance amount, press（2）Middle extracting method
Extract simultaneously parallel determination；
（9）Serviceability test：The content of 6 kinds of organic acid compositions in the chromatographic column determination sample of different brands is respectively adopted；
（10）Test limit and quantitative limit：When signal to noise ratio is 3, neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4- are measuredO- two coffees
Coffee acyl group quininic acid, 3,5-O- DCQ, 4,5-OThe test limit of-DCQ is respectively
1.165th, 0.973,1.088,0.945,0.994 and 1.315ng；When signal to noise ratio is 10, neochlorogenic acid, chlorogenic acid, hidden is measured
Chlorogenic acid, 3,4-O- DCQ, 3,5-O- DCQ, 4,5-O- DCQ
Quantitative limit is respectively 3.882,3.243,3.628,3.149,3.312 and 4.383ng；
（11）Using chlorogenic acid as internal standard, the methods commented are surveyed using one to its organic acid composition more and are measured, by investigating not
With the instrument of brand, pillar, different mobile phase ratios etc., using chlorogenic acid as internal reference thing, the relative correction factor of 5 kinds of compositions is established
（f k/s ）, calculate the amount of each composition.
The beneficial effects of the invention are as follows：
The present invention establishes the survey of one kind one and comments method more（QMSA methods）It is new to determine 6 kinds of organic acid compositions in Mussaenda hirsutula simultaneously
Chlorogenic acid, Cryptochlorogenic acid, chlorogenic acid, 3,5-O- DCQ, 3,4-O- DCQ, 4,5-O- two
The method of caffeoyl quinic acid, the organic acid composition of this method selection Mussaenda hirsutula carry out assay for index,
Precision height, favorable reproducibility, stability is good, the rate of recovery is high, measurement result is accurate, can effectively control the matter of Mussaenda hirsutula
Amount.The perfect quality inspection standard of Mussaenda hirsutula of the present invention, is established for the foundation of Mussaenda hirsutula quality standard
Basis.
Embodiment
The present invention is introduced in order to more detailed, with reference to embodiment, the present invention will be further described.
Embodiment, content are as follows：
（1）Chromatographic condition：Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as stream
Dynamic phase B, gradient elution is carried out by table 1；The μ L of sample size 10,35 DEG C of column temperature, the mL/min of flow velocity 1.0, the nm of Detection wavelength 328.
The Mussaenda hirsutula gradient elution table of table 1
（2）Extract sample：Take sample powder（Cross No. four sieves）About 0.5 g, it is accurately weighed, put in conical flask with cover, precision adds
The mL of 50% methanol 20, weighed weight, it is ultrasonically treated（The W of power 320, the kHz of frequency 40）30 min, take out, let cool, then it is weighed heavy
Amount, the weight of less loss is supplied with 50% methanol, is shaken up, and is filtered, is taken filtrate, produce.
（3）The preparation of 6 kinds of reference substance storing solutions：
Neochlorogenic acid（5- caffeoyl quinic acids）--- precision weighs the mg of neochlorogenic acid reference substance 9.52（Chengdu Man Sitesheng
＆ Chengdu Inst. of Biology, Chinese Academy of Sciences of thing Science and Technology Ltd. develops, lot number：MUST-16021806, content is with 99.37%
Meter）, put in 50 mL measuring bottles, add methanol to make dissolving and be diluted to scale, shake up, standby, Stock concentrations are 0.1892 mg/
mL；
Chlorogenic acid（3- caffeoyl quinic acids）--- precision weighs the mg of chlorogenic acid reference substance 10.07（Zhong Jian institutes provide, lot number
110753-201415, content is in terms of 96.2%）, put in 10 mL measuring bottles, add methanol to make dissolving and be diluted to scale, shake up, it is standby
With Stock concentrations are 0.9687 mg/mL；
Cryptochlorogenic acid（4- caffeoyl quinic acids）--- precision weighs the mg of Cryptochlorogenic acid reference substance 11.17（Chengdu Man Sitesheng
＆ Chengdu Inst. of Biology, Chinese Academy of Sciences of thing Science and Technology Ltd. develops, lot number：MUST-16022403, content is with 99.07%
Meter）, put in 10 mL measuring bottles, add methanol to make dissolving and be diluted to scale, shake up, standby, Stock concentrations are 1.1066 mg/
mL；
3,4-O- DCQ --- precision weighs 3,4-OThe mg of-DCQ reference substance 11.01（Into
All ＆ Chengdu Inst. of Biology, Chinese Academy of Sciences of Man Site bio tech ltd develops, lot number：MUST-15081411, contain
Amount is in terms of 99.05%）, put in 10 mL measuring bottles, add methanol to make dissolving and be diluted to scale, shake up, standby, Stock concentrations are
1.0905 mg/mL；
3,5-O- DCQ --- precision weighs 3,5-OThe mg of-DCQ reference substance 10.71（In
Examine institute to provide, lot number 111782-201405, content is in terms of 92.0%）, put in 10 mL measuring bottles, add methanol to make dissolving and be diluted to
Scale, shake up, standby, Stock concentrations are 0.9853 mg/mL；
4,5-O- DCQ --- precision weighs 4,5-OThe mg of-DCQ reference substance 10.19（In
Examine institute to provide, lot number 111894-201102, content is in terms of 94.1%）, put in 10 mL measuring bottles, add methanol to make dissolving and be diluted to
Scale, shake up, standby, Stock concentrations are 0.9589 mg/mL.
（4）The preparation of 4 kinds of mixed reference substance solutions：
Mixed reference substance solution is 1. --- and above-mentioned each 1.00 mL of 6 kinds of reference substance storing solutions is drawn respectively and is put in 10 mL measuring bottles, is added
Methanol shakes up, produced, the mixed solution is containing neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4- to scaleO- two coffee acyl Kuis
Peaceful acid, 3,5-O- DCQ, 4,5-OThe concentration of-DCQ be respectively 18.92 μ g/mL,
96.87 μg/mL、110.66 μg/mL、109.05 μg/mL、98.53 μg/mL、95.89 μg/mL；
Mixed reference substance solution is 2. --- and precision draws mixed reference substance solution, and 1. 1.00 mL are put in 5 mL measuring bottles, add methanol extremely
Scale, shake up, produce, the mixed solution is containing neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4-O- DCQ, 3,
5-O- DCQ, 4,5-OThe concentration of-DCQ is respectively 3.784 μ g/mL, 19.375 μ g/
mL、22.132 μg/mL、21.811 μg/mL、19.706 μg/mL、19.178 μg/mL；
Mixed reference substance solution is 3. --- and precision draws mixed reference substance solution, and 2. 1.00 mL are put in 5 mL measuring bottles, add methanol extremely
Scale, shake up, produce, the mixed solution is containing neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4-O- DCQ, 3,
5-O- DCQ, 4,5-OThe concentration of-DCQ is respectively 0.7568 μ g/mL, 3.8749 μ
g/mL、4.4264 μg/mL、4.3622 μg/mL、3.9413 μg/mL、0.8355 μg/mL；
Mixed reference substance solution is 4. --- and precision draws mixed reference substance solution, and 3. 1.00 mL are put in 5 mL measuring bottles, add methanol extremely
Scale, shake up, produce, the mixed solution is containing neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4-O- DCQ, 3,
5-O- DCQ, 4,5-OThe concentration of-DCQ is respectively 0.1514 μ g/mL, 0.7750 μ
g/mL、0.8853 μg/mL、0.8724 μg/mL、0.7883 μg/mL、0.7671 μg/mL。
（5）It is linear to investigate：Above-mentioned 6 kinds of reference substance storing solutions are pressed（1）Middle μ L of chromatographic condition sample introduction 10,4 kinds of reference substances mix
Close solution 1., 2., 3., 4. distinguish the μ L of sample introduction 10, respectively as 5 points of linear concentration from high to low, with the sample introduction of reference substance
Amount（ng）For abscissa, peak area is ordinate, draws standard curve, the results showed that：
When neochlorogenic acid reference substance sample size is in the range of 1.514~1892 ng, sample size is with peak area in good linear pass
System, regression equation are：Y=912643X+1446.6, r=1.0000；
When chlorogenic acid reference substance sample size is in the range of 7.750~9687 ng, sample size is with peak area in good linear pass
System, regression equation are：Y=1E+06X+12376, r=1.0000；
When Cryptochlorogenic acid reference substance sample size is in the range of 8.853~11066 ng, sample size is with peak area in well linear
Relation, regression equation are：Y=848861X+5659.4, r=0.9995；
3,4-OWhen-DCQ reference substance sample size is in the range of 8.724~10905 ng, sample size and peak face
Product is in good linear relationship, and regression equation is：Y=1E+06X+8317.8, r=1.0000；
3,5-OWhen-DCQ reference substance sample size is in the range of 7.883~9853 ng, sample size and peak area
In good linear relationship, regression equation is：Y=1E+06X+12123, r=1.0000；
4,5-OWhen-DCQ reference substance sample size is in the range of 7.671~9589 ng, sample size and peak area
In good linear relationship, regression equation is：Y=1E+06X+3881, r=1.0000.
（6）Precision test：Take with a need testing solution（Numbering 012046-1）, press（1）Middle chromatographic condition, it is continuous to survey
It is fixed 6 times.9 kinds of component contents of 6 measure are as shown in table 2（N=6）, show that the precision of instrument is good.
The precision measurement result table of table 2
（7）Reappearance test：Take with a collection of test sample（Numbering 012046）Powder about 0.5g, it is accurately weighed, press（2）Middle extraction side
6 parts of method extraction and parallel determination, 9 kinds of component contents of 6 parts of measure are as shown in table 3（N=6）, the results showed that the reappearance of this law
Preferably.
The reappearance measurement result table of table 3
Remarks：1.~first part to the 6th part of repeated sampling is 6. represented, average is the average of six parts of assay results, and unit is equal
For mg/g.
（8）Accuracy test：Take with a collection of test sample（Numbering 012046）Powder about 0.25g, it is accurately weighed, totally 12 parts,
The content of the 9 kinds of compositions calculated by precision result is separately added into three levels（50%、100%、150%）Reference substance amount,
Press（2）Middle extracting method extraction and parallel determination, 9 kinds of compositions sample-adding recovery result of 12 parts of measure are as shown in table 4（N=4）, table
The degree of accuracy of bright this law is preferable.
The sample-adding recovery measurement result table of table 4
Remarks：Sample-adding result is the average of 4 parts of parallel determinations above（n=4）, unit is %.
（9）Serviceability test：The chromatographic column of different brands is respectively adopted（KromasiL、TechMateC18ST、
AgiLentTCC18, three root chromatogram column specifications are：5 μm, 4.6mm × 250mm）Determination sample（012046）In 6 kinds of organic acids
The content of constituents, the results are shown in Table 5, show this law good tolerance.
5 different chromatographic columns of table investigate result（Content unit is mg/g）
（10）Test limit and quantitative limit：When signal to noise ratio is 3, neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4- are measuredO- two coffees
Coffee acyl group quininic acid, 3,5-O- DCQ, 4,5-OThe test limit of-DCQ is respectively
1.165th, 0.973,1.088,0.945,0.994 and 1.315ng；When signal to noise ratio is 10, neochlorogenic acid, chlorogenic acid, hidden is measured
Chlorogenic acid, 3,4-O- DCQ, 3,5-O- DCQ, 4,5-O- DCQ
Quantitative limit is respectively 3.882,3.243,3.628,3.149,3.312 and 4.383ng.
（11）Mussaenda hirsutula one surveys the foundation for commenting method more：
Using chlorogenic acid as internal standard, the methods commented are surveyed using one to its organic acid composition more and are measured, by investigating different product
The instrument of board, pillar, different mobile phase ratios etc., using chlorogenic acid as internal reference thing, establish the relative correction factor of 5 kinds of compositions
（f k/s ）, the amount of each composition is calculated, the results are shown in Table 6~7.
6 Radix Mussaendae of table, 6 kinds of composition relative retention times and correction factor
Table 7 one is surveyed comments method and Standard reference experimental data value contrast table more
As known from Table 7, RD values ＜ 3%, a survey comments method and Standard reference that one can be used to survey and comment method to carry out more without significant difference
Assay.
Claims (1)
1. the multicomponent content assaying method of Mussaenda hirsutula, it is characterised in that methods described includes herein below：
（1）Chromatographic condition：Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as stream
Dynamic phase B, carries out gradient elution, elution time 70min；The μ L of sample size 10,35 DEG C, flow velocity 1.0mL/min of column temperature, Detection wavelength
328nm；
（2）Extract sample：Take sample powder（Cross No. four sieves）About 0.5g, it is accurately weighed, put in conical flask with cover, precision adds
50% methanol 20mL, weighed weight, 30min is ultrasonically treated, takes out, let cool, then weighed weight, the weight of less loss is supplied with 50% methanol
Amount, shakes up, and filters, takes filtrate, produce；
（3）Prepare neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4-O- DCQ, 3,5-O- two coffee acyl Kuis
Peaceful acid, 4,5-O- DCQ reference substance storing solution；
（4）4 kinds of mixed reference substance solutions of different linear concentrations are prepared with above-mentioned 6 kinds of reference substance storing solutions；
（5）It is linear to investigate：Above-mentioned 6 kinds of reference substance storing solutions is molten by above-mentioned μ L of chromatographic condition sample introduction 10,4 kinds of reference substances mixing
Liquid distinguishes the μ L of sample introduction 10, respectively as 5 points of linear concentration from high to low, with the sample size of reference substance（ng）For abscissa,
Peak area is ordinate, draws standard curve；
（6）Precision test：Take with a need testing solution, by above-mentioned chromatographic condition, METHOD FOR CONTINUOUS DETERMINATION 6 times；
（7）Reappearance test：Take with a collection of test sample powder about 0.5g, it is accurately weighed, press（2）Middle extracting method extraction is simultaneously parallel
6 parts of measure；
（8）Accuracy test：Take with a collection of test sample powder about 0.25g, it is accurately weighed, totally 12 parts, calculated by precision result
The content of 6 kinds of compositions out is separately added into three levels（50%、100%、150%）Reference substance amount, carried by said extracted method
Take simultaneously parallel determination；
（9）Serviceability test：The content of 6 kinds of organic acid compositions in the chromatographic column determination sample of different brands is respectively adopted；
（10）Test limit and quantitative limit：When signal to noise ratio is 3, neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 3,4- are measuredO- two coffees
Coffee acyl group quininic acid, 3,5-O- DCQ, 4,5-OThe test limit of-DCQ is respectively
1.165th, 0.973,1.088,0.945,0.994 and 1.315ng；When signal to noise ratio is 10, neochlorogenic acid, chlorogenic acid, hidden is measured
Chlorogenic acid, 3,4-O- DCQ, 3,5-O- DCQ, 4,5-O- DCQ
Quantitative limit is respectively 3.882,3.243,3.628,3.149,3.312 and 4.383ng；
（11）Measure：Using chlorogenic acid as internal standard, the methods commented are surveyed using one to its organic acid composition more and are measured, by examining
Instrument, pillar, different mobile phase ratios of different brands etc. are examined, using chlorogenic acid as internal reference thing, establishes the relative correction of 5 kinds of compositions
The factor（f k/s ）, calculate the content of each composition.
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| CN109709259A (en) * | 2019-02-01 | 2019-05-03 | 贵州医科大学 | A kind of discrimination method of Radix Mussaendae |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109709259A (en) * | 2019-02-01 | 2019-05-03 | 贵州医科大学 | A kind of discrimination method of Radix Mussaendae |
| CN109709259B (en) * | 2019-02-01 | 2020-07-07 | 贵州医科大学 | Method for identifying mussaenda pubescens |
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