CN106248813A - The content assaying method of the Radix Aucklandiae, Cortex Magnoliae Officinalis in a kind of SHUGAN WAN - Google Patents
The content assaying method of the Radix Aucklandiae, Cortex Magnoliae Officinalis in a kind of SHUGAN WAN Download PDFInfo
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- CN106248813A CN106248813A CN201610527133.3A CN201610527133A CN106248813A CN 106248813 A CN106248813 A CN 106248813A CN 201610527133 A CN201610527133 A CN 201610527133A CN 106248813 A CN106248813 A CN 106248813A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention provides the content assaying method of the Radix Aucklandiae, Cortex Magnoliae Officinalis in a kind of SHUGAN WAN.The method uses honokiol, Radix Aucklandiae oxylactone, dehydrocostuslactone, the content of four kinds of compositions of magnolol in hplc simultaneous determination SHUGAN WAN, and method is sensitive, accurately, can be used for the quality control of SHUGAN WAN.
Description
Technical field
The present invention relates to the content assaying method of the Radix Aucklandiae, Cortex Magnoliae Officinalis in a kind of SHUGAN WAN, belong to technical field of Chinese medicines.Specifically
, relate to honokiol in a kind of SHUGAN WAN, Radix Aucklandiae oxylactone, dehydrocostuslactone, four kinds of compositions of magnolol measure simultaneously
Content assaying method.
Background technology
SHUGAN WAN is qi-regulating prescription, has soothing liver-QI stomach function regulating, effect of regulating QI to relieve pain.Cure mainly stagnation of QI due to depression of the liver, costa sternales distension, gastral cavilty
Pain, noisy vomiting, belch pantothenic acid.This medicine is by Fructus Toosendan, vinegar Rhizoma Corydalis, Rhizoma Wenyujin Concisum, the Radix Paeoniae Alba (wine stir-fry), Lignum Aquilariae Resinatum, Fructus Aurantii
(stir-fry), the Radix Aucklandiae, Fructus Amomi, Pericarpium Citri Reticulatae, Fructus Amomi Rotundus core, Poria, Cortex Magnoliae Officinalis(processed with ginger), Cinnabaris composition, drug effect is clear and definite.SHUGAN WAN (big honeyed pills) standard is
Early being embodied in " Chinese Pharmacopoeia " version in 1985, increase water-honeyed pill specification in versions in 2000, within 2005, version increases watered pill specification, and right
Standard is revised, the same working standard of inspection content, increases small honey pill specification in version the second enlarged editions in 2010, and inspection content is same
" Chinese Pharmacopoeia 2005 version, revises.This medicine working standard is " Chinese Pharmacopoeia " version in 2015, describes this medicine
Prescription, preparation method and method of quality control.Flavour of a drug contained by this medicine are many, in the assay item in " Chinese Pharmacopoeia " version in 2015,
Sample, after complicated processing procedure, has only carried out assay, the Radix Aucklandiae, Cortex Magnoliae Officinalis in SHUGAN WAN prescription to peoniflorin in the Radix Paeoniae Alba
In honokiol, Radix Aucklandiae oxylactone, dehydrocostuslactone, magnolol be not measured.
Summary of the invention
Object of the present invention is to provide the Radix Aucklandiae, the content assaying method of Cortex Magnoliae Officinalis in a kind of SHUGAN WAN of mensuration simultaneously, this
Invention uses honokiol, Radix Aucklandiae oxylactone, dehydrocostuslactone, magnolol in the high effective liquid chromatography for measuring Radix Aucklandiae, Cortex Magnoliae Officinalis
Four kinds of component contents.
Content assaying method of the present invention uses high performance liquid chromatography, and the method is as follows:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；Flowing be mutually acetonitrile-
0.05% phosphoric acid, volume ratio is 47: 53；Detection wavelength is 230-240nm, and flow velocity is 1ml/min, and number of theoretical plate presses honokiol
Peak calculates should be not less than 2000-5000；
The preparation of reference substance solution takes honokiol reference substance, Radix Aucklandiae oxylactone reference substance, dehydrocostuslactone comparison
Product, magnolol reference substance are appropriate, accurately weighed, add methanol and make every 1ml mixed solution respectively containing 10,12,25,20 μ g,
Obtain；
The preparation of need testing solution takes SHUGAN WAN water-honeyed pill, finely ground, takes 0.8g, accurately weighed；Or take small honey pill or sweet greatly
Ball, shreds, and takes 0.8g, accurately weighed, puts in tool plug conical flask, accurate addition methanol 25ml, close plug, and weighed weight heats back
Flow 30-120 minute, take out, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, i.e.
?；
Algoscopy precision respectively draws reference substance solution 10 μ l and need testing solution 20 μ l, injects chromatograph of liquid, surveys
Fixed, to obtain final product；
In content assaying method of the present invention, detection wavelength is preferably 230nm；Number of theoretical plate is preferably based on honokiol peak
Calculation should be not less than 3000；The time that is heated to reflux in test sample preparation is preferably 90 minutes.
In order to verify the mensuration stability of content method, accuracy, specificity and system suitability, method has been carried out with
Lower Method validation is tested, to guarantee that this content assaying method can be as the quality control of SHUGAN WAN.
1 instrument and reagent
1.1 instrument
High performance liquid chromatograph: Waters e2695；Chromatographic column: Thermo Accliam (5 μm, 4.6mm × 250mm)；
Electronic balance: Mettler AE163；Mettler AE240；Ultra-pure water instrument: Integral 10 (Millipore).
1.2 reagents and reagent
Reference substance: honokiol, Radix Aucklandiae oxylactone, dehydrocostuslactone, magnolol (are purchased from Chinese food drug assay
Academy；Prepare negative control medical material: Fructus Toosendan, vinegar Rhizoma Corydalis, the Radix Paeoniae Alba (wine stir-fry), Rhizoma Wenyujin Concisum, the Radix Aucklandiae, Lignum Aquilariae Resinatum, Fructus Amomi Rotundus core,
Fructus Amomi, Cortex Magnoliae Officinalis(processed with ginger), Pericarpium Citri Reticulatae, Fructus Aurantii (parched), Poria, Cinnabaris are purchased from market, through Hebei province's drug inspection academy's professor of pharmacy's section
Ji Ping is accredited as certified products.
Reagent: methanol, acetonitrile (chromatographically pure, Merck company)；Water is ultra-pure water；Other reagent are analytical pure.
2 methods determine
The determination of 2.1 chromatographic conditions
2.1.1 the selection measuring wavelength weighs honokiol, Radix Aucklandiae oxylactone, dehydrocostuslactone, magnolol pair respectively
Appropriate according to product, it is configured to the reference substance solution of suitable concentration with methanol solution, carries out spectral scan at 200nm～400nm.Result
Showing, Magnolol and Honokiol has end absorption maximum, dehydrocostuslactone and Radix Aucklandiae oxylactone to exist near 210nm wavelength
Absorption maximum is had near 220-240nm wavelength.Under 200～220nm wavelength, sample is measured, result composition to be measured with
Impurity peaks separates poor, therefore selects composition to be measured response relatively strong and the 230-240nm of free from admixture peak interference is as measuring wavelength,
Under this wavelength, each composition to be measured separates with impurity peaks well, and negative noiseless, wherein 230nm effect is best.
The selection of the phase that 2.1.2 flows
Sample prescription taste of Chinese medicine is many, complicated component, and impurity is many, and the composition that character is close is many, and flowing is made organic with methanol
Phase time, fails to obtain good separation, investigates and the investigation of chromatographic condition in conjunction with extracting method, and final selection acetonitrile is as organic
Phase, according to polarity and the appearance time of each composition to be measured, is set to acetonitrile-0.05% phosphoric acid solution by ratio, and volume ratio is 47:
53, separate good.
The preparation of 2.2 reference substance solution
The solvent of need testing solution is methanol solution, therefore the dilution of reference substance solution selects methanol solution.
The preparation method of 2.3 need testing solutions
The preparation of need testing solution takes SHUGAN WAN water-honeyed pill, finely ground, takes 0.8g, accurately weighed；Or take small honey pill or sweet greatly
Ball, shreds, and takes 0.8g, accurately weighed.Put in tool plug conical flask, accurate addition methanol 25ml, close plug, weighed weight, heats back
Flow 90 minutes, take out, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product.
The investigation of 2.4 extracting modes
Taking same batch sample (big honeyed pills, lot number: 4015087) appropriate, be divided into two parts, precision adds methanol 25ml respectively,
A supersound process (power 400W, frequency 40kHz) 90 minutes, another part are heated to reflux 90 minutes, survey by drafting chromatographic condition
Fixed.Result shows to be heated to reflux more slightly higher than supersound process extraction ratio, therefore selects the extracting mode being heated to reflux, and is shown in Table 1.
The assay result of the different extracting mode of table 1
The investigation of 2.5 extraction times
Take same batch sample (big honeyed pills, lot number: 4015087) appropriate, be heated to reflux respectively 30min, 60min, 90min,
120min, measures by drafting chromatographic condition.It is relatively big that result shows that Radix Aucklandiae content is affected by return time, and Cortex Magnoliae Officinalis content results base
This indifference, for guaranteeing to extract completely, selects to be heated to reflux 90min, is shown in Table 2.
The assay result of table 2 different extraction time
The investigation of 2.6 Extraction solvent
Take same batch sample (big honeyed pills, lot number: 4015087) appropriate, respectively with 50%, 70% methanol solution, methanol,
Diluted Alcohol is Extraction solvent, measures by drafting chromatographic condition.Result shows with methanol as Extraction solvent, and measured composition to be measured contains
Measure higher, select methanol as Extraction solvent with reference to official method, be shown in Table 3.
The assay result of table 3 different solvents
3 methodological studies
3.1 specificity tests
Prepare without the Radix Aucklandiae, the negative sample of Cortex Magnoliae Officinalis respectively in prescription ratio and preparation method, by the preparation side of need testing solution
Method prepares negative sample solution, measures by text method, and analyzes chromatographic peak purity in diode array detector.Result table
Bright, in side, other flavour of a drug do not disturb honokiol, Radix Aucklandiae oxylactone, dehydrocostuslactone, the mensuration of magnolol, and chromatogram is shown in figure
1。
3.2 linear relationships are investigated
Precision weighs each reference substance in right amount respectively, preparation mixing reference substance solution (honokiol concentration 0.02004mg/ml,
Radix Aucklandiae oxylactone concentration 0.02493mg/ml, dehydrocostuslactone concentration 0.05146mg/ml, magnolol concentration 0.03978mg/
Ml), precision measures mixing reference substance solution 2ml and puts in 10ml measuring bottle, adds methanol dilution to scale, shakes up, obtain mixing reference substance
Solution II.Precision measures mixing reference substance solution II 5 μ l, 10 μ l, and mixing reference substance solution 5 μ l, 10 μ l, 20 μ l inject liquid phase
Chromatograph, is measured by the chromatographic condition drafted, and records peak area.With reference substance sample size (μ g) as abscissa, the peak of reference substance
Area integral value is vertical coordinate, carries out linear regression.Result honokiol, Radix Aucklandiae oxylactone, dehydrocostuslactone, magnolol line
Property equation is respectively y=3.724 × 106x-5.70 × 104, y=1.650 × 106x-1.55 × 104, y=1.131 × 106x-
2.16 × 104, y=5.315 × 106x-3.98 × 104；The range of linearity be respectively 0.02004～0.4008 μ g, 0.02493～
0.4986 μ g, 0.05146～1.0291 μ g, 0.03978～0.7955 μ g；Correlation coefficient is 0.9999, shows composition to be measured
Linear relationship is good.Experimental result is shown in Table 4.
The each component linear relation to be measured of table 4 investigates result
3.3 replica test
Take same batch sample (big honeyed pills, lot number: 4015087), shred, weigh 0.64g, 0.8g, 0.96g each three respectively
Part, accurately weighed, make need testing solution by need testing solution preparation method, measure by text method.Result shows, this method
Repeatability is good (the results are shown in Table 5).
Table 5 replica test result
3.4 recovery test
Take the sample (big honeyed pills, lot number: 4015087 content are shown in repeatability result) of known content, shred, take 9 parts, every part
About 0.4g, accurately weighed, every three parts is one group, and the mixing of basic, normal, high three concentration of the most accurate addition solution preparation is right
According to product solution 25ml, low concentration (honokiol: 0.001203mg ml-1；Radix Aucklandiae oxylactone: 0.002493mg ml-1；Dehydrogenation
Constuslactone: 0.004117mg ml-1；Magnolol: 0.003182mg ml-1)；Intermediate concentration (honokiol:
0.001503mg·ml-1；Radix Aucklandiae oxylactone: 0.003116mg ml-1；Dehydrocostuslactone: 0.005146mg ml-1；Cortex Magnoliae Officinalis
Phenol: 0.003978mg ml-1)；High concentration (honokiol: 0.001804mg ml-1；Radix Aucklandiae oxylactone: 0.003740mg
ml-1；Dehydrocostuslactone: 0.006175mg ml-1；Magnolol: 0.004773mg ml-1).By need testing solution preparation
Lower section legal system available test sample solution, is measured by text method, calculates the response rate.Result shows that this method response rate is good.It is shown in Table 6
～9.
Table 6 honokiol recovery test result
Table 7 Radix Aucklandiae oxylactone recovery test result
Table 8 dehydrocostuslactone recovery test result
Table 9 magnolol recovery test result
3.5 stability test
Taking same need testing solution, started in 0 hour to measure, later certain interval of time measures once, records peak area.
Result RSD is respectively 1.2%, 1.5%, 1.2%, 0.7%, shows that need testing solution at least stably (be shown in 24 hours by result
Table 10).
Table 10 need testing solution stability test result
4 sample determinations are formulated with limit
By drafting method, 34 batch sample are tested, the results are shown in Table 11.
Table 11 Radix Aucklandiae, Cortex Magnoliae Officinalis assay result (part)
Accompanying drawing explanation
Fig. 1: reference substance (A), sample (B), Radix Aucklandiae negative sample (C), Cortex Magnoliae Officinalis negative sample (D), in chromatogram, 1 is and thick
Piao's phenol, 2 is Radix Aucklandiae oxylactone, and 3 is dehydrocostuslactone, and 4 is magnolol).
Detailed description of the invention
Embodiment 1
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；Flowing be mutually acetonitrile-
0.05% phosphoric acid, volume ratio is 47: 53；Detection wavelength is 230nm；Flow velocity is 1ml/min, and number of theoretical plate is based on honokiol peak
Calculation should be not less than 3000；
The preparation of reference substance solution takes honokiol reference substance, Radix Aucklandiae oxylactone reference substance, dehydrocostuslactone comparison
Product, magnolol reference substance are appropriate, accurately weighed, add methanol and make every 1ml mixed solution respectively containing 10,12,25,20 μ g,
Obtain；
The preparation of need testing solution takes SHUGAN WAN water-honeyed pill, finely ground, takes 0.8g, accurately weighed；Or take small honey pill or sweet greatly
Ball, shreds, and takes 0.8g, accurately weighed.Put in tool plug conical flask, accurate addition methanol 25ml, close plug, weighed weight, heats back
Flow 90 minutes, take out, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；
Algoscopy precision respectively draws reference substance solution 10 μ l and need testing solution 20 μ l, injects chromatograph of liquid, surveys
Fixed, to obtain final product.
Embodiment 2
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；Flowing be mutually acetonitrile-
0.05% phosphoric acid, volume ratio is 47: 53；Detection wavelength is 240nm；Flow velocity is 1ml/min, and number of theoretical plate is based on honokiol peak
Calculation should be not less than 4000；
The preparation of reference substance solution takes honokiol reference substance, Radix Aucklandiae oxylactone reference substance, dehydrocostuslactone comparison
Product, magnolol reference substance are appropriate, accurately weighed, add methanol and make every 1ml mixed solution respectively containing 10,12,25,20 μ g,
Obtain；
The preparation of need testing solution takes SHUGAN WAN water-honeyed pill, finely ground, takes 0.8g, accurately weighed；Or take small honey pill or sweet greatly
Ball, shreds, and takes 0.8g, accurately weighed.Put in tool plug conical flask, accurate addition methanol 25ml, close plug, weighed weight, heats back
Flow 30 minutes, take out, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；
Algoscopy precision respectively draws reference substance solution 10 μ l and need testing solution 20 μ l, injects chromatograph of liquid, surveys
Fixed, to obtain final product.
Embodiment 3
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；Flowing be mutually acetonitrile-
0.05% phosphoric acid, volume ratio is 47: 53；Detection wavelength is 235nm；Flow velocity is 1ml/min, and number of theoretical plate is based on honokiol peak
Calculation should be not less than 2000；
The preparation of reference substance solution takes honokiol reference substance, Radix Aucklandiae oxylactone reference substance, dehydrocostuslactone comparison
Product, magnolol reference substance are appropriate, accurately weighed, add methanol and make every 1ml mixed solution respectively containing 10,12,25,20 μ g,
Obtain；
The preparation of need testing solution takes SHUGAN WAN big honeyed pills, shreds, and takes 0.8g, accurately weighed；Or take small honey pill or sweet greatly
Ball, shreds, and takes 0.8g, accurately weighed.Put in tool plug conical flask, accurate addition methanol 25ml, close plug, weighed weight, heats back
Flow 60 minutes, take out, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；
Algoscopy precision respectively draws reference substance solution 10 μ l and need testing solution 20 μ l, injects chromatograph of liquid, surveys
Fixed, to obtain final product.
Embodiment 4
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；Flowing be mutually acetonitrile-
0.05% phosphoric acid, volume ratio is 47: 53；Detection wavelength is 230nm；Flow velocity is 1ml/min, and number of theoretical plate is based on honokiol peak
Calculation should be not less than 5000；
The preparation of reference substance solution takes honokiol reference substance, Radix Aucklandiae oxylactone reference substance, dehydrocostuslactone comparison
Product, magnolol reference substance are appropriate, accurately weighed, add methanol and make every 1ml mixed solution respectively containing 10,12,25,20 μ g,
Obtain；
The preparation of need testing solution takes SHUGAN WAN concentrated pill, finely ground, takes 0.8g, accurately weighed；Or take small honey pill or sweet greatly
Ball, shreds, and takes 0.8g, accurately weighed.Put in tool plug conical flask, accurate addition methanol 25ml, close plug, weighed weight, heats back
Flow 120 minutes, take out, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；
Algoscopy precision respectively draws reference substance solution 10 μ l and need testing solution 20 μ l, injects chromatograph of liquid, surveys
Fixed, to obtain final product.
Above-described embodiment all carries out Method validation according to pharmacopoeial requirements, result presentation method accurately and reliably, sensitive, special
Belonging to, indices all meets the requirement of quality control.
Claims (5)
1. a content assaying method for the Radix Aucklandiae, Cortex Magnoliae Officinalis in SHUGAN WAN, the method uses high performance liquid chromatography, it is characterised in that
This content assaying method is as follows:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；Flowing be mutually acetonitrile-
0.05% phosphoric acid, volume ratio is 47: 53；Detection wavelength is 230-240nm, and flow velocity is 1ml/min, and number of theoretical plate presses honokiol
Peak calculates should be not less than 2000-5000；
The preparation of reference substance solution takes honokiol reference substance, Radix Aucklandiae oxylactone reference substance, dehydrocostuslactone reference substance, thickness
Piao's phenol reference substance is appropriate, accurately weighed, adds methanol and makes every 1ml mixed solution respectively containing 10,12,25,20 μ g, to obtain final product；
The preparation of need testing solution takes SHUGAN WAN water-honeyed pill, finely ground, takes 0.8g, accurately weighed；Or take small honey pill or big honeyed pills,
Shred, take 0.8g, accurately weighed.Put in tool plug conical flask, accurate addition methanol 25ml, close plug, weighed weight, it is heated to reflux
30-120 minute, take out, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；
Algoscopy precision respectively draws reference substance solution 10 μ l and need testing solution 20 μ l, injects chromatograph of liquid, measures, i.e.
?.
Content assaying method the most according to claim 1, it is characterised in that described detection wavelength is 230nm.
Content assaying method the most according to claim 1, it is characterised in that described number of theoretical plate is for based on honokiol peak
Calculation should be not less than 3000.
Content assaying method the most according to claim 1, it is characterised in that described test sample is heated to reflux the time when preparing
It it is 90 minutes.
5. according to the content assaying method described in any one of claim 1-4, it is characterised in that this content assaying method is as follows:
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；Flowing be mutually acetonitrile-
0.05% phosphoric acid, volume ratio is 47: 53；Detection wavelength is 230nm；Flow velocity is 1ml/min, and number of theoretical plate is based on honokiol peak
Calculation should be not less than 3000；
The preparation of reference substance solution takes honokiol reference substance, Radix Aucklandiae oxylactone reference substance, dehydrocostuslactone reference substance, thickness
Piao's phenol reference substance is appropriate, accurately weighed, adds methanol and makes every 1ml mixed solution respectively containing 10,12,25,20 μ g, to obtain final product；
The preparation of need testing solution takes SHUGAN WAN water-honeyed pill, finely ground, takes 0.8g, accurately weighed；Or take small honey pill or big honeyed pills,
Shred, take 0.8g, accurately weighed；Put in tool plug conical flask, accurate addition methanol 25ml, close plug, weighed weight, it is heated to reflux 90
Minute, take out, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, take subsequent filtrate, to obtain final product；
Algoscopy precision respectively draws reference substance solution 10 μ l and need testing solution 20 μ l, injects chromatograph of liquid, measures, i.e.
?.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101559192A (en) * | 2008-04-15 | 2009-10-21 | 北京亚东生物制药有限公司 | Traditional Chinese medicine granular formulation for warming stomach and regulating middle warmer and quality control method thereof |
| CN102008704A (en) * | 2007-05-23 | 2011-04-13 | 北京亚东生物制药有限公司 | Detection method for composition having middle-warming stomach harmonizing function |
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2016
- 2016-06-29 CN CN201610527133.3A patent/CN106248813A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008704A (en) * | 2007-05-23 | 2011-04-13 | 北京亚东生物制药有限公司 | Detection method for composition having middle-warming stomach harmonizing function |
| CN101559192A (en) * | 2008-04-15 | 2009-10-21 | 北京亚东生物制药有限公司 | Traditional Chinese medicine granular formulation for warming stomach and regulating middle warmer and quality control method thereof |
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| Title |
|---|
| VADAPARTHI, P. R. RAO等: "Estimation of Costunolide and Dehydrocostus Lactone in Saussurea lappa and its Polyherbal Formulations followed by their Stability Studies Using HPLC-DAD", 《PHARMACOGNOSY MAGAZINE》 * |
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Application publication date: 20161221 |