CN109001310A - A kind of detection method of rhodiola kirilowii Regel - Google Patents
A kind of detection method of rhodiola kirilowii Regel Download PDFInfo
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- CN109001310A CN109001310A CN201810675150.0A CN201810675150A CN109001310A CN 109001310 A CN109001310 A CN 109001310A CN 201810675150 A CN201810675150 A CN 201810675150A CN 109001310 A CN109001310 A CN 109001310A
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- G—PHYSICS
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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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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- G01N2030/027—Liquid chromatography
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- 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
- G01N2030/062—Preparation extracting sample from raw material
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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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- G01N2030/146—Preparation by elimination of some components using membranes
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Abstract
The invention discloses a kind of detection methods of rhodiola kirilowii Regel, which is characterized in that this method includes that rhodioside, tyrosol reference substance is taken to prepare to obtain reference substance solution；Rhodiola kirilowii Regel test solution and the reference substance solution is taken to be detected.For measuring method of the invention using rhodioside and the good tyrosol of bioactivity as content Con trolling index, the preparation method of test article of this method is simple, detection process is efficient, testing result is stable, accurate.
Description
Technical field
The present invention relates to technical field of analytical chemistry, in particular to a kind of quality determining method of rhodiola kirilowii Regel.
Background technique
Rhodiola kirilowii Regel has the effect of activating microcirculation and removing stasis medicinal, coronary circulation-promoting pain-relieving, wherein its capsule is by single medicinal material rhodiola kirilowii Regel
Rhodiola kirilowii (Regel.) Maxim is extracted, is refined.There are one for the existing quality standard of rhodiola kirilowii Regel capsule
Fixed limitation cannot more comprehensively control the requirement of this quality, and preparation method of test article is complicated, so that detection process
Not enough efficiently.It is therefore desirable to further study preparation method of test article and chromatographic condition, new method of quality control is developed,
The measuring method of measurement rhodioside and tyrosol content while finally establishing quick, easy, stable, accurate.
Summary of the invention
In view of this, purport of the present invention proposes a kind of detection method of rhodiola kirilowii Regel medicinal material, which is characterized in that this method packet
It includes, rhodioside, tyrosol reference substance is taken to prepare to obtain reference substance solution；Take rhodiola kirilowii Regel preparation test solution and described
Reference substance solution is detected.By comparing, qualitative with retention time, with peak area quantification, rhodiola kirilowii Regel can be obtained
Qualitative and quantitative information.Wherein, which can be the crude drug or its medicine materical crude slice or be that its is any of Chinese medicine
Optional preparation.
Preferably, the reference substance solution prepare it is specific as follows: take rhodioside, tyrosol reference substance appropriate, precision claim
It is fixed, add methanol that the mixed reference substance solution that every 1mL is respectively 712.3 μ g, 181.4 μ g containing rhodioside, tyrosol is made.
Further, when the rhodiola kirilowii Regel medicinal material is selected from capsule, the preparation of the test solution using reflux or
Ultrasonic method extracts sample, and preferably using the aqueous solution of methanol, ethyl alcohol, methanol or ethyl alcohol as Extraction solvent, Extraction solvent can be with
It is mixing or its mixing aqueous solvent of methanol/ethanol.
Specifically, the preparation method of the test solution is to take rhodiola kirilowii Regel capsule, is mixed, finely ground, accurately weighed,
Methanol, weighed weight, reflux, then weighed weight is added, the weight of less loss is supplied with methanol, the test sample for preparing 20mg/ml is molten
Liquid.
Specifically, the time that the circumfluence method extracts sample is greater than 60 minutes.The preparation method of the test solution is also
It may include filtration step, preferably through 0.22 μm of filtering with microporous membrane, take subsequent filtrate, obtain test solution.
Further, the chromatographic condition of the detection are as follows: use C18Chromatographic column, it is molten containing buffering using acetonitrile as mobile phase A
The water phase of liquid is Mobile phase B, using gradient elution mode, gradient elution program are as follows: 0~5min, 5%A；5~18min, 5%~
25%A；18~30min, 25%~75%A；30-40 DEG C of column temperature, flow velocity 0.8-1.5mL/min, Detection wavelength 270-
280nm.Sample volume can be 5 μ L.
Specifically, the buffer solution be selected from one of weak acid and its salt, weak base and its salt, Multiple Weak Acid and its salt or
It is a variety of；It is preferred that trifluoroacetic acid, formic acid, ammonium formate, acetic acid, sodium acetate, ammonium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, di(2-ethylhexyl)phosphate
One of hydrogen potassium, citric acid, sodium citrate, glycine, hydrochloric acid, phthalic acid are a variety of；More preferable 0.3% glacial acetic acid.
Specifically, the C18Chromatographic column is Kromasil 100-3.5C18Chromatographic column, Agilent Zorbax SB-C18Color
Compose column, Phenomenex Gemini C18Chromatographic column, Thermo syncronis C18Chromatographic column, Agilent 5TC-C18Chromatography
Column, Kromasil 100-5C18Chromatographic column, Waters symmetry C18Chromatographic column or Phenomenex Luna C18Chromatography
Column；It is preferred that Kromasil 100-3.5C18Chromatographic column.
Preferably, the column temperature is 37 DEG C.
Further, the flow velocity is 1.2mL/min.
Preferably, the Detection wavelength is 275nm.
In the measuring method of rhodiola kirilowii Regel proposed by the present invention, which is characterized in that the measuring method is using HPLC
Method simultaneously in rhodiola kirilowii Regel rhodioside and tyrosol be measured.
The method of the present invention has good precision, linear relationship, stability, repeatability, and the rate of recovery is high, and durability is good
It is good；This method meets the requirement for more comprehensively controlling this quality, and energy measures root of kirilow rhodiola while quick, easy, stable, accurate
Glycosides and tyrosol content can be used as the effective ways of the quality of evaluation rhodiola kirilowii Regel medicinal material.
Detailed description of the invention
Fig. 1 is the corresponding HPLC chromatogram of elution program I, II, III；
Fig. 2 is ethyl alcohol-ultrasonic extraction HPLC figure；
Fig. 3 is methanol-ultrasonic extraction HPLC figure；
Fig. 4 is ethyl alcohol-refluxing extraction HPLC figure；
Fig. 5 is methanol-refluxing extraction HPLC figure；
Fig. 6 is that the HPLC of 50% methanol-refluxing extraction schemes；
Fig. 7 is that the HPLC of 50% methanol-ultrasonic extraction schemes；
Fig. 8 is the HPLC figure that 50% alcohol reflux extracts；
Fig. 9 is the HPLC figure that 50% EtOH Sonicate extracts；
Figure 10 is rhodioside, the linear result figure of tyrosol.
Specific embodiment
The invention discloses a kind of detection method of rhodiola kirilowii Regel capsule, those skilled in the art can be used for reference in this paper
Hold, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.
Except particularly pointing out, drug used, reagent, instrument in rhodiola kirilowii Regel capsule measuring method provided by the invention
It is bought by conven-tional channels or market.
1 instrument and reagent
1260 high performance liquid chromatograph of Agilent, Agilent company of the U.S.；
1200 high performance liquid chromatograph of Agilent, Agilent company of the U.S.；
Waters 2695-2998 high performance liquid chromatograph, Waters, US；
Chromatographic column: Kromasil (250 × 4.6mm, 3.5 μm)；
MILLIPORE Milli-Q Century pure water meter, Millipore company of the U.S.；
BSA224S-CW type electronic analytical balance, German Sartorius company；
Mettler Toledo X6 type electronic analytical balance, Mettler company of Switzerland；
Mettler AL204 type electronic analytical balance, Mettler company of Switzerland；
500-DB type ultrasonic washing instrument, Kunshan Ultrasonic Instruments Co., Ltd.；
HH- digital display thermostat water bath, Changzhou Guo Yu instrument manufacturing Co., Ltd；
Rhodioside reference substance (National Institute for Food and Drugs Control, content is in terms of 99.4%), tyrosol reference substance (China
Food and medicine examines and determine research institute, and content is in terms of 100%)；
Rhodiola kirilowii Regel capsule (Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov)；
Acetonitrile (the upper biochemical Co., Ltd of starfish, chromatographically pure), remaining reagent are that analysis is pure.
2 chromatographic conditions
The selection of 2.1 Detection wavelengths
Take test solution, rhodioside and tyrosol the reference substance solution full wavelength scanner within the scope of 200-400nm, red scape
Its glycosides and tyrosol have near 220nm and 275nm compared with strong absworption peak, and at 275nm, baseline is more steady, and impurity is less,
Therefore it is preferred that select the Detection wavelength that 275nm is rhodioside and tyrosol.
2.2 mobile phases it is preferred
With Kromasil 100-3.5C18(4.6 × 150mm, 3.5 μm) column is stationary phase, using gradient elution mode, with
Acetonitrile is as mobile phase A, and 0.3% glacial acetic acid is as Mobile phase B, and by being investigated to different elution programs, (elution program is shown in
Table 1~3), the results showed that I separating effect of elution program is preferable, the results are shown in Table 4, sees Fig. 1.
Table 1, gradient elution program I
Table 2, gradient elution program II
Table 3, gradient elution program III
Table 4, three kind of gradient elution program separation effect comparison
3 system suitabilities
3.1 reference substance solutions are prepared
Take rhodioside, tyrosol reference substance appropriate, it is accurately weighed, add methanol that every 1mL is made and distinguishes containing rhodioside, tyrosol
For the mixed reference substance solution of 712.3 μ g, 181.4 μ g.
The preparation of 3.2 test solutions
This product under content uniformity item is taken, is mixed, is taken in right amount, it is finely ground, about 0.5g is taken, it is accurately weighed, it sets in round-bottomed flask,
Methanol 25mL is added in precision, and weighed weight flows back 60 minutes, then weighed weight, and the weight of less loss is supplied with methanol, filters
(0.22 μm of miillpore filter), takes subsequent filtrate, as test solution.
3.3 measurement
Precision draws rhodioside, 5 μ l of tyrosol reference substance solution, injects high performance liquid chromatograph, continuously repeats sample introduction 6
Secondary, measurement calculates the relative standard deviation of peak area measurement value；Precision draws 5 μ l of test solution, injects high performance liquid chromatography
Instrument continuously repeats sample introduction 6 times, and measurement calculates peak area, theoretical cam curve, separating degree and the tailing factor of component to be measured
RSD% the results are shown in Table 5-6.
Table 5, reference substance solution measurement result
Table 6, test solution measurement result
3.4 result
Under the above conditions, rhodioside, tyrosol and neighbouring chromatographic peak separating degree are all larger than 1.5 in sample chromatogram, reach
To baseline separation, symmetrical factor is between 0.88~1.00, and peak area repeatability relative standard deviation is respectively less than 2%, theoretical tower
Plate number is all larger than 60000；The result shows that this method instrument system applicability is good.
4 durabilities are investigated
It is carried out according to chromatographic condition under " 2 chromatography condition " item and sample solution preparation method and reference substance solution resistance to
It is investigated with property.As a result as follows:
4.1 column temperatures are investigated
When fixed flow rate is 1.2ml/min, investigating when column temperature is 35 DEG C, 37 DEG C, 42 DEG C influences the content of ingredient to be measured,
It the results are shown in Table 7, the results showed that when column temperature is 35 DEG C, 37 DEG C, 42 DEG C, the separation of rhodioside and tyrosol is preferable at 37 DEG C.
Table 7, different column temperatures are investigated
Further investigating when column temperature is 36 DEG C, 37 DEG C influences the content of ingredient to be measured, the results showed that the smaller change of column temperature
Influence to component content result to be measured is smaller, therefore 35 DEG C -42 DEG C can be used to separate, preferably column temperature be 36-37 DEG C, more preferable 37
℃.It the results are shown in Table 9.
4.2 flow velocitys are investigated
Fixed 37 DEG C of column temperature, investigates the influence different in flow rate to separating effect.As a result 0.8mL/min, 1.1mL/min,
When 1.3mL/min, rhodioside cannot be separated preferably with tyrosol, the results are shown in Table 8.
Table 8, investigation result different in flow rate
Fixed 37 DEG C of column temperature continues to investigate when flow velocity is respectively 1.18mL/min, 1.20mL/min, 1.22mL/min and treat
The content for surveying ingredient influences, and preferable separation can be obtained, the results showed that flow velocity is smaller to be changed to component content result to be measured without aobvious
Difference is write, therefore selects flow velocity 1.2mL/min, the results are shown in Table 9.
4.3 chromatographic columns are investigated
Chromatographic column is investigated, preferably Kromasil (150 × 4.6mm, 3.5 μm) chromatographic column separating degree is preferable, final choice color
Spectrum column is Kromasil 100-3.5.
4.4 instruments are investigated
Stationary chromatographic condition, chromatographic column investigate the influence that different instruments measure component content to be measured.It the results are shown in Table 9.
The result shows that: Agilent 1200, Agilent 1260, Waters2695-2998 are used equally for ingredient to be measured to contain
It is fixed to measure.
4.5 result
In summary it investigates condition and calculates rhodioside and tyrosol content results and be shown in Table 9, as follows:
Table 9, durability investigate result
4.6 conclusion
Investigate result according to durability: investigate result according to durability: when flow velocity is preferably 1.20mL/min, column temperature is preferred
It does not make significant difference within the scope of 37 DEG C ± 1 DEG C to component content result to be measured；At fixed 37 DEG C of column temperature, flow velocity 1.20mL/min ±
It does not make significant difference when within the scope of 0.02mL/min to component content to be measured.The result shows that: the flow velocity and column temperature after preferably are smaller
To component content result to be measured without significant difference when change.To sum up, preferred rhodioside, tyrosol measurement chromatographic condition are determined
Are as follows: with Kromasil 100-3.5C18(4.6 × 150mm, 3.5 μm) column is that stationary phase is made using gradient elution mode with acetonitrile
For mobile phase A, 0.3% glacial acetic acid is as Mobile phase B, and 37 DEG C of column temperature, flow velocity 1.2mL/min, 5 μ L of sample volume, Detection wavelength is
275nm, elution program I.
The preparation method of 5 test solutions is investigated
In order to extract rhodioside, tyrosol in preparation completely, to extracting mode, Extraction solvent, extraction time, extraction
Solvent volume is investigated respectively.
5.1 Extraction solvents and extracting method are investigated
Test sample is taken, it is finely ground, about 2g is taken, it is accurately weighed, it totally 8 parts, sets in stuffed conical flask, it is accurate respectively that 50% first is added
Alcohol, methanol, 50% ethyl alcohol, each 50ml of ethyl alcohol, close plug, weighed weight flow back 1 hour (90 DEG C) or are ultrasonically treated respectively 60 points
Clock is let cool, then weighed weight, and the weight of less loss is supplied with corresponding reagent, is shaken up, filtration, take subsequent filtrate to get.It the results are shown in Table
10。
Table 10, Extraction solvent investigate result
The result shows that: 50% methanol, methanol, 50% ethyl alcohol, ethyl alcohol are Extraction solvent, and HPLC schemes (Fig. 2-9) and shows methanol
The peak shape for extracting sample chromatogram peak is good, and 50% methanol and 50% ethyl alcohol extraction impurity are more, and baseline is more crude；Reflux and ultrasound ratio
Compared with reflux is slightly better than ultrasonic extraction, and preferably methanol eddy extracts.
5.2 extraction times were investigated
The test sample under content uniformity item is taken, is mixed, is taken in right amount, it is finely ground, 4 parts, every part of about 1.0g are weighed respectively, and precision claims
It is fixed, it sets in round-bottomed flask, precision is added methanol 50mL, weighed weight, and return time is respectively 20 minutes, 40 minutes, 60 minutes,
It 80 minutes, lets cool, then weighed weight, the weight of less loss is supplied with methanol, is shaken up, filter (0.22 μm of miillpore filter), take continuous filter
Liquid carries out assay as test solution, and measurement result is shown in Table 11.
Table 11, rhodioside, tyrosol extraction time investigate result (mg/g)
The result shows that 60 minutes i.e. extractable complete, therefore the selective extraction time is 60 minutes.
5.3 post processing extraction solvent consumptions are investigated
The test sample under content uniformity item is taken, is mixed, is taken in right amount, it is finely ground, 3 parts, every part of about 1.0g are weighed respectively, and precision claims
It is fixed, it sets in round-bottomed flask, accurate respectively that methanol 25mL, 50mL and 100mL is added, weighed weight flows back 60 minutes, lets cool, then
Weighed weight is supplied the weight of less loss with methanol, is shaken up, and filters (0.22 μm of miillpore filter), takes subsequent filtrate, molten as test sample
Liquid carries out assay, the results are shown in Table 12.
Table 12, solvent usage investigate result (mg/g)
The result shows that: sample weighting amount and Extraction solvent amount ratio are respectively 1:25, rhodioside and tyrosol when 1:50,1:100
Content is without significant difference, therefore sample weighting amount and Extraction solvent amount ratio are 1:25,1:50,1:100, in view of scape red in test sample
Its glycosides content is higher, and to obtain suitable peak area, therefore it is preferred that sample weighting amount and Extraction solvent amount ratio are 1:50, i.e. 0.5g is for examination
Methanol 25ml is added in product to extract.
5.4 conclusion
It according to above-mentioned experimental result, determines that preparation method of test article is as follows: taking this product under content uniformity item, mix, take
In right amount, finely ground, about 0.5g is taken, it is accurately weighed, it sets in round-bottomed flask, methanol 25mL is added in precision, and weighed weight flows back 60 points
Clock, then weighed weight supply the weight of less loss with methanol, filter (0.22 μm of miillpore filter), take subsequent filtrate, molten as test sample
Liquid.
6. methodology validation is investigated
6.1 specificities are investigated
6.1.1 the preparation of reference substance solution
Take rhodioside, tyrosol reference substance appropriate, it is accurately weighed, add methanol that every 1mL is made and distinguishes containing rhodioside, tyrosol
For the mixed reference substance solution of 712.3 μ g, 181.4 μ g.
6.1.2 the preparation of test solution
This product under content uniformity item is taken, is mixed, is taken in right amount, it is finely ground, about 0.5g is taken, it is accurately weighed, it sets in round-bottomed flask,
Methanol 25mL is added in precision, and weighed weight flows back 60 minutes, then weighed weight, and the weight of less loss is supplied with methanol, filters
(0.22 μm of miillpore filter), takes subsequent filtrate, as test solution.
6.1.3 Peak homogeneity
Purity verifying, test sample color are carried out to rhodioside, tyrosol in sample chromatogram with diode array detector
Rhodioside, tyrosol chromatographic peak Reinheitszahl are all larger than threshold value in spectrum, meet the requirements.
6.2 linear relationships are investigated
It is appropriate that precision weighs rhodioside, tyrosol reference substance, add methanol be made mixed standard solution (rhodioside:
2471.442 μ g/mL, tyrosol: 599.560 μ g/mL).Draw respectively above mixed standard solution 0.1mL, 0.2mL, 0.5mL,
1.0mL, 1.2mL, 2.0mL add methanol dilution to be settled to scale in 5mL measuring bottle, shake up up to the curve of 6 mixing reference substances
Concentration.Above-mentioned 5 μ L of solution is drawn respectively, injects high performance liquid chromatograph, and measurement the results are shown in Table 13~14, Figure 10.With peak area
Average value is ordinate (Y), and reference substance concentration (μ g/mL) is abscissa (X), draws standard curve and obtains regression equation:
Rhodioside: Y=1204X-3055.9；R=0.9999
Tyrosol: Y=2736.6X-1237.8；R=0.9999
Table 13, linear relationship investigation-rhodioside
Table 14, linear relationship investigation-tyrosol
The result shows that rhodioside is in good linear relationship, junket between 49.429~988.577 μ g/mL in concentration
Alcohol is in good linear relationship between 11.991~239.824 μ g/mL in concentration.
6.3 precision test
Take basic, normal, high three various concentrations mixing contrast solution (rhodioside concentration successively are as follows: 49.429,
247.144,988.577 μ g/mL, tyrosol concentration is successively are as follows: 11.991,59.956,239.824 μ g/mL and repeated 1 test sample
Solution continuous sample introduction 6 times respectively, the results are shown in Table 15, peak area RSD is below 2%, the results showed that instrument precision is good.
Table 15, precision result
6.4 repetitive test
Test sample is taken, 6 parts of test solutions are made by sample solution preparation method, measures in accordance with the law, external standard is respectively adopted
One point method and calibration curve method calculate sample result, and two kinds of calculation method results are without significant difference.It the results are shown in Table 16.The result shows that
This method repeatability is good.
Table 16, repeated result (mg/g)
6.5 stability test
Take same test solution respectively at rhodioside, junket in 0,2,4,6,8,18,25 hour sample introduction, test solution
The RSD of alcohol peak area is respectively 1.48%, 1.28%, shows that test solution is stablized in 25 hours.It the results are shown in Table 17.
Table 17, stability result (peak area)
6.6 recovery test
It is appropriate that precision weighs rhodioside, tyrosol reference substance, add methanol be made mixed standard solution (rhodioside:
2471.442 μ g/mL, tyrosol: 599.56 μ g/mL).
Take test sample (the one point external standard method calculating: Determination of Salidroside 10.076mg/g, tyrosol content under content uniformity item
2.041mg/g；Calibration curve method calculates: Determination of Salidroside 10.174mg/g, tyrosol content 2.062mg/g), it mixes, takes suitable
Amount, it is finely ground, weigh 9 parts, every part of about 0.5g respectively, it is accurately weighed, it sets in round-bottomed flask, wherein the sample that number is 1,2,3 is every
Part is separately added into mixed reference substance solution 0.5mL, and every part of sample of number 4,5,6 is separately added into mixed reference substance solution
1.0mL, every part of sample of number 7,8,9 are separately added into mixed reference substance solution 1.5mL, reference substance solution to be mixed with for examination
After product are sufficiently mixed, solvent evaporated, every part accurate again to be added methanol 25mL, and weighed weight flows back 60 minutes, lets cool to room temperature,
The weight that less loss is supplied with methanol, shakes up, and filters (0.22 μm of miillpore filter), takes subsequent filtrate, as test solution, contained
It is fixed to measure, and one point external standard method is respectively adopted and calibration curve method calculates content.It the results are shown in Table 18.
Table 18, one point external standard method calculate sample recovery rate result
As a result: two kinds of calculation method rate of recovery of rhodioside and tyrosol are between 94.9%~105.8%, and RSD% is not
Greater than 2.1%.The result shows that this method rate of recovery is good.
7. multiple batches of sample survey
5 batches of test samples are taken, test solution are made by 3.2 lower sample solution preparation methods, according to 4.6 lower chromatographies
Condition carries out assay to rhodioside, tyrosol.It the results are shown in Table 19.
Table 19,5 batch sample testing results
Primary standard measuring method: it is numbered referring to rhodiola kirilowii Regel capsule standard: WS3-1047 (Z-271) -2008Z.
Primary standard measurement result and rebuilding method measurement result relatively have certain deviation, and analysis reason may be since original is marked
Quasi- preparation method of test article is complex, and measurement result is caused to be slightly below rebuilding method, the results are shown in Table 20.
Table 20, Determination of Salidroside difference Comparison between detecting methods (mg/g)
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of detection method of rhodiola kirilowii Regel medicinal material, which is characterized in that this method includes taking rhodioside, tyrosol control
Product are prepared to obtain reference substance solution；The test solution and the reference substance solution for taking rhodiola kirilowii Regel medicinal material are detected.
2. detection method according to claim 1, which is characterized in that the rhodiola kirilowii Regel medicinal material is capsule preparations, institute
The preparation for stating test solution extracts sample using reflux or ultrasonic method, is to mention with the aqueous solution of methanol, ethyl alcohol, methanol or ethyl alcohol
Take solvent.
3. detection method according to claim 1 or 2, which is characterized in that the chromatographic condition of the detection are as follows: use C18Color
Column is composed, using acetonitrile as mobile phase A, the water phase containing buffer solution is Mobile phase B, using gradient elution mode, gradient elution journey
Sequence are as follows: 0~5min, 5%A；5~18min, 5%~25%A；18~30min, 25%~75%A；30-40 DEG C of column temperature, flow velocity
0.8-1.5mL/min, Detection wavelength 270-280nm.
4. detection method according to claim 3, which is characterized in that the buffer solution is selected from weak acid and its salt, weak base
And its one of salt, Multiple Weak Acid and its salt or a variety of；It is preferred that trifluoroacetic acid, formic acid, ammonium formate, acetic acid, sodium acetate, acetic acid
Ammonium, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, citric acid, sodium citrate, glycine, hydrochloric acid, in phthalic acid
It is one or more；More preferable 0.3% glacial acetic acid.
5. detection method according to claim 3, which is characterized in that the C18Chromatographic column is Kromasil 100-3.5C18
Chromatographic column, Agilent Zorbax SB-C18Chromatographic column, Phenomenex Gemini C18Chromatographic column, Thermo
syncronis C18Chromatographic column, Agilent 5TC-C18Chromatographic column, Kromasil 100-5C18Chromatographic column, Waters
symmetry C18Chromatographic column or Phenomenex Luna C18Chromatographic column；It is preferred that Kromasil 100-3.5C18Chromatographic column.
6. measuring method according to claim 3, which is characterized in that the column temperature is 37 DEG C.
7. detection method according to claim 3, which is characterized in that the flow velocity is 1.18-1.22mL/min, preferably
1.2mL/min。
8. detection method according to claim 3, which is characterized in that the Detection wavelength is 275nm.
9. detection method according to claim 2, which is characterized in that the time that the circumfluence method extracts sample is greater than 60 points
Clock.
10. a kind of measuring method of rhodiola kirilowii Regel capsule, which is characterized in that the measuring method is right simultaneously using HPLC method
Rhodioside and tyrosol in rhodiola kirilowii Regel capsule are measured.
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| SU1725109A1 (en) * | 1989-10-03 | 1992-04-07 | Научно-исследовательский институт фармакологии Томского научного центра АМН СССР | Determination of polar organic compounds by liquid chromatography method |
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