CN105277642A - Method for simultaneously quantitatively determining content of multiple phenols compositions in gastrodia elata Bl. - Google Patents
Method for simultaneously quantitatively determining content of multiple phenols compositions in gastrodia elata Bl. Download PDFInfo
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- CN105277642A CN105277642A CN201410252945.2A CN201410252945A CN105277642A CN 105277642 A CN105277642 A CN 105277642A CN 201410252945 A CN201410252945 A CN 201410252945A CN 105277642 A CN105277642 A CN 105277642A
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Abstract
The invention relates to a content determining new method for simultaneously determining content of gastrodin, p-hydroxybenzylalcohol, parishin B, parishin C, parishin and other phenols compositions in gastrodia elata Bl.. The main steps comprise performing separation by utilizing rapid high performance liquid chromatography employing methanol or acetonitrile and an acid water with the volume percentage of 0.1%-0.5% as a mobile phase, determining the content of the target compounds by utilizing a high-sensitivity fluorescence detection process, and controlling the excitation wavelength to be 215-235 nm and the emission wavelength to be 295-335 nm. The method is high in sensitivity, good in precision and short in analysis time, and is applicable to rapid sensitive determination on phenols compositions in gastrodia elata Bl. medicinal material and extracts.
Description
Technical field
The present invention relates to the detection field of Chinese medicinal material, be specifically related to the detection method of Chinese medicinal material rhizoma Gastrodiae.
Background technology
Rhizoma Gastrodiae has another name called rhizoma gastrodiae, DINGFENGCAO, solely shakes sesame, is the dry tuber of the orchid family Gastrodia plant rhizoma Gastrodiae (GastrodiaelataBl.).Shennong's Herbal before bimillennium is classified as top grade, the effect of have suppressing hyperactive liver for calming endogenous wind, removing obstruction in channels to relieve pain, and has the title of " the refreshing medicine controlling wind ".Cure mainly the diseases such as headache is dizzy, extremity numbness, epilepsy clonus.Gastrodin is the main active of rhizoma Gastrodiae, control the quality of Rhizoma Gastrodiae with the content of Gastrodin in 2010 editions Chinese Pharmacopoeias, its content assaying method is high performance liquid chromatography, be Stationary liquid with octadecyl base silane bonded silica gel, with acetonitrile-0.05% phosphoric acid solution (3: 97) for mobile phase, determined wavelength 220nm.Recent study shows, rhizoma Gastrodiae active component, except Gastrodin, also exists the multiple phenols component such as p-Hydroxybenzylalcohol, parishinB, parishinC and parishin.As the people such as Wang Ke have been found that parishin constituents has certain protective effect (rhizoma Gastrodiae Pai Lixin extract is preparing the application in brain-protection drugs, Chinese Patent Application No. 201210230669.0) to the neural cell injury of cerebral ischemia re-pouring injured, N-methyl-D-aspartate induction and Closed cerebral trauma.Therefore, be expected to control rhizoma Gastrodiae quality more comprehensively to phenols Simultaneously test multiple in rhizoma Gastrodiae.And the method for document or patent only can realize measuring (Liu Yuhong to the content of 1 ~ 4 kind of phenols component in rhizoma Gastrodiae or total Gastrodin at present, Pharmaceutical Analysis magazine, 2010,30 (1): 30-32) (Liu Yuhong, Chinese patent drug, 2012,34 (1): 182-184) (Liu Zhi, Chinese Pharmaceutical Journal, 2012,40 (5): 380-383), the reflection rhizoma Gastrodiae being difficult to objective reality plays whole active components of drug action in vivo, and method sensitivity is low.In addition, report is had no for the multiple phenols component in Simultaneously test rhizoma Gastrodiae.Therefore the multiple phenols component in a kind of simple to operate, quick, highly sensitive method Simultaneously test rhizoma Gastrodiae is needed.Based on above reason, this invention exploits a kind of simultaneously to the high performance liquid chromatography-fluorescence method of Gastrodin in Gastrodia eleta Bl., p-Hydroxybenzylalcohol, the multiple phenols component such as parishinB, parishinC and parishin.
Summary of the invention
The object of this invention is to provide the content assaying method of multiple phenols component in rhizoma Gastrodiae, described assay method utilizes high performance liquid chromatography-fluorescence method to measure Gastrodin in Gastrodia eleta Bl., p-Hydroxybenzylalcohol, parishinB, parishinC and parishin content simultaneously, and carried out linear relationship investigation, stability test, precision test, replica test and application of sample recovery test are verified method, prove that this analysis method is accurate, reliably.Described method is simple, quick, sensitive, and the quality assessment that can be rhizoma Gastrodiae provides foundation.
For achieving the above object, the technical solution used in the present invention:
A content assaying method for multiple phenols component in Simultaneous Determination rhizoma Gastrodiae, mainly comprises following three steps, it is characterized in that:
1) reference standards and the preparation for examination product to be tested solution: rhizoma Gastrodiae phenols reference standards and Rhizoma Gastrodiae extract to be measured being dissolved in respectively volumetric concentration is in the methyl alcohol of 0-8% or the aqueous solution of acetonitrile, preparation reference standards mixed solution and need testing solution to be measured;
2) with high performance liquid chromatography-fluorescence method for means carry out the quantitative test of reference standards mixed solution and need testing solution composition to be measured respectively;
Analyzed by the reference standards mixed solution of the rhizoma Gastrodiae phenolic compound to a series of different quality concentration, obtain the retention time of each rhizoma Gastrodiae phenolic compound in reference standards mixed solution and peak area corresponding to each rhizoma Gastrodiae phenolic compound different quality concentration;
By the analysis to need testing solution to be measured, obtain the retention time of rhizoma Gastrodiae phenolic compound and the peak area of correspondence in Rhizoma Gastrodiae extract to be measured;
3) content of phenols component in external standard method rhizoma Gastrodiae;
Analyzed by the reference standards mixed solution of the rhizoma Gastrodiae phenolic compound to a series of different quality concentration, using the known quality concentration of rhizoma Gastrodiae phenolic compound and corresponding peak area as transverse and longitudinal coordinate, set up each known rhizoma Gastrodiae phenolic compound typical curve;
With the retention time of rhizoma Gastrodiae phenolic compound each in reference standards mixed solution for benchmark, treating rhizoma Gastrodiae phenolic compound in observation nettle extract carries out qualitative, the peak area of rhizoma Gastrodiae phenolic compound in Rhizoma Gastrodiae extract to be measured after qualitative is substituted in its corresponding rhizoma Gastrodiae phenolic compound typical curve, it is carried out quantitatively.
Step 1) described in the preparation method of Rhizoma Gastrodiae extract be solvent extraction method, wherein preferred alcohol-water extraction is got, described extracting method can be in refluxing extraction, ultrasonic extraction, microwave radiation exaraction any one, preferred preparation method is: Rhizoma Gastrodiae 5-10 volumetric concentration 70% alcohol-water refluxing extraction doubly 2 ~ 3 times, extract merges concentrated, with suitable quantity of water dispersion, and the extraction into ethyl acetate of 1-2 times of volume 2-4 time, the water layer obtained merges concentrated, to obtain final product.Prepare the reference substance mixed solution such as Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin with 8% methanol-water for solvent and prepare Rhizoma Gastrodiae extract need testing solution, wherein said rhizoma Gastrodiae phenols component mainly comprises Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin.
Step 2) described in the chromatographic condition of method be: C18 liquid-phase chromatographic column; Mobile phase is methyl alcohol or acetonitrile (A)-volume fraction 0.1 ~ 0.5% aqueous acid (B); Gradient program wash-out; Column temperature: 40 ~ 60 DEG C; Flow velocity: 0.25 ~ 0.35ml/min; Excitation wavelength 215nm ~ 235nm, emission wavelength 295 ~ 335nm; Sample size 1 ~ 20 μ l; Wherein optimum condition is mobile phase is methyl alcohol-aqueous acid, and sour preferable formic acid, its volume fraction is 0.5%.Column temperature is 50 DEG C, and flow velocity is 0.35ml/min, and sample size is 1 μ l.Excitation wavelength is 225nm, and emission wavelength is 310nm.
Step 3) in compound concentration be the Gastrodin of 5,25,50,250,500,2500ng/ml, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin mixing reference substance standard solution carry out the mensuration of typical curve.
Known rhizoma Gastrodiae contains Gastrodin, p-Hydroxybenzylalcohol, parahydroxyben-zaldehyde, parishin constituents, especially parishinB, parishinC and parishin are high-visible in chromatogram, peak area is comparatively large, parishinB, parishinC and parishin can be used for the quality control of rhizoma Gastrodiae as index components.Adopt content assaying method of the present invention to measure five compositions representative in rhizoma Gastrodiae, make the composition of rhizoma Gastrodiae definitely, quality control is more strict, for the quality control of rhizoma Gastrodiae provides data.
Content assaying method of the present invention has the following advantages:
1) the present invention adopts high performance liquid chromatography-fluorescence method, at excitation wavelength 225nm, the assay while of can disposablely carrying out the Gastrodin in Rhizoma Gastrodiae or extract, p-Hydroxybenzylalcohol, the multiple phenols component such as parishinB, parishinC and parishin under emission wavelength 310nm.Improve detection efficiency.The method yet there are no bibliographical information.
2) the present invention adopts the means of high performance liquid chromatography-fluorescence, and compared with conventional efficient liquid phase-ultraviolet detection method, sensitivity can bring up to ng/ml by ug/ml, improves 1000 times.
3) the method is highly sensitive, precision good, analysis time is short, can be used for quick, the sensitive determination of phenols component in Rhizoma Gastrodiae, Rhizoma Gastrodiae extract.
Accompanying drawing explanation
Fig. 1 Gastrodin, p-Hydroxybenzylalcohol, the full excitation wavelength of parishinB, parishinC and parishin200-290nm absorb figure.
Fig. 2 Gastrodin, p-Hydroxybenzylalcohol, the full emission wavelength of parishinB, parishinC and parishin280-500nm absorb figure.
Fig. 3 mobile phase is followed successively by from top to bottom: acetonitrile-water, acetonitrile-0.5% formic acid water, methanol-water, methyl alcohol-0.5% formic acid water, column temperature 50 DEG C, flow velocity 0.35ml/min, chromatographic column is the chromatogram of AgilentZORBAXSB-C18 (3.0 × 100mm, 1.8 μm).
Fig. 4 column temperature is followed successively by 40 DEG C from top to bottom, 50 DEG C, 60 DEG C, and mobile phase is methyl alcohol-0.5% formic acid water, flow velocity 0.35ml/min, and chromatographic column is the chromatogram of AgilentZORBAXSB-C18 (3.0 × 100mm, 1.8 μm).
Fig. 5 flow velocity is followed successively by 0.25ml/min from top to bottom, 0.30ml/min, 0.35ml/min, and mobile phase is methyl alcohol-0.5% formic acid water, column temperature 50 DEG C, and chromatographic column is the chromatogram of AgilentZORBAXSB-C18 (3.0 × 100mm, 1.8 μm).
The mixing reference substance of Fig. 6 Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin and the chromatogram of Rhizoma Gastrodiae extract, wherein Fig. 6 A is from left to right respectively Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin.
Embodiment
The instrument used in following examples and reagent:
Agilent1200 type highly effective liquid phase chromatographic system, is equipped with G1367C automatic sampler system, G1316B type column oven, G1365C type Variable wavelength UV detector and G1321 variable wavelength fluorescence detector (Agilent company of the U.S.); BSA-224S electronic balance (German Sartorius); Chromatographic column AgilentZORBAXSB-C18 (3.0 × 100mm, 1.8 μm).
Gastrodin reference substance, p-Hydroxybenzylalcohol reference substance, parishinB reference substance, parishinC reference substance and parishin reference substance; Above reference substance is all separated from this laboratory, and purity >98% meets quantitative requirement.
The preparation method of Rhizoma Gastrodiae extract is solvent extraction method, wherein preferred alcohol-water extraction is got, described extracting method can be in refluxing extraction, ultrasonic extraction, microwave radiation exaraction any one, preferred preparation method is: Rhizoma Gastrodiae 5-10 volumetric concentration 70% alcohol-water refluxing extraction doubly 2 ~ 3 times, extract merges concentrated, with suitable quantity of water dispersion, and the extraction into ethyl acetate of 1-2 times of volume 2-4 time, the water layer obtained merges concentrated, to obtain final product.
Methyl alcohol (chromatographically pure), water is deionized water, and it is pure that all the other are analysis.
The optimization of embodiment 1 excitation wavelength, emission wavelength and chromatographic condition
The screening of excitation-emission full wavelength scanner
Get Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin reference substance respectively in right amount, accurately weighed, dissolve with 8% methanol-water and be diluted to the reference substance stock solution that concentration is 1mg/ml.
A certain amount of 5 kinds of reference substance stock solutions of accurate absorption, add 8% methanol-water and are diluted to the mixing reference substance solution confession condition optimizing analysis that concentration is 100ng/ml;
To concentration be the Gastrodin of 100ng/ml, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin reference substance mixed solution carry out full excitation wavelength (200 ~ 290nm) scanning, find that these five compositions have absorption maximum at 225nm place, as shown in Figure 1.Determining under excitation wavelength, to concentration be the Gastrodin of 100ng/ml, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin reference substance mixed solution carry out full emission wavelength (280 ~ 500nm) scanning, find that these five compositions have absorption maximum at 310nm place, as shown in Figure 2.
Investigated the flow phase system such as acetonitrile-water, acetonitrile-0.5% formic acid water, methanol-water, methyl alcohol-0.5% formic acid water, result as shown in Figure 3, compares through test, finally determines the assay completing above five compositions with methyl alcohol-0.5% formic acid water gradient elution.
Investigated 40 DEG C, 50 DEG C, 60 DEG C different column temperatures, result as shown in Figure 4; And 0.25,0.3,0.35ml/min different in flow rate on the impact of each component separating, result is as shown in Figure 5; Find 50 DEG C, flow velocity be 0.35ml/min time be separated best results.
The content detection of Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin in embodiment 2 Rhizoma Gastrodiae extract
Chromatographic condition
Chromatographic column is AgilentZORBAXSB-C18 (3.0 × 100mm, 1.8 μm), and mobile phase is methyl alcohol (A)-0.5% formic acid solution (B), elution program: 0-5min, A linearly rises to 40%, 5-12min from 8%, and A maintains 40%; Column temperature: 50 DEG C; Flow velocity: 0.35ml/min; Excitation wavelength 225nm, emission wavelength 310nm; Sample size 10 μ l.Get need testing solution, sample introduction analysis under above-mentioned chromatographic condition, in sample, the degree of separation of each component to be measured and adjacent peak is all greater than 1, and chromatogram as shown in Figure 6.
Prepared by reference substance solution
Get Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin reference substance respectively in right amount, accurately weighed, dissolve with 8% methanol-water and be diluted to the reference substance stock solution that concentration is 1mg/ml.
A certain amount of 5 kinds of reference substance stock solutions of accurate absorption, add 8% methanol-water and are diluted to the mixing reference substance solution that concentration is 100ng/ml;
Prepared by test sample
Take Rhizoma Gastrodiae extract (lot number 132100) 10mg, accurately weighed, put in 50ml measuring bottle, add 8% methanol-water and dissolve and constant volume, shake up, both.
Linear relationship is tested
Precision measures Gastrodin, p-Hydroxybenzylalcohol, parishinB, in the appropriate and 10ml volumetric flask of parishinC and parishin reference substance stock solution, with 8% methanol-water dilution constant volume, shake up, both reference substance solution must be mixed, the doubly dilution of reference substance solution 8% methanol-water will be mixed again, constant volume, both the mixing reference substance solution of 6 concentration needed for typical curve had been obtained, shake up, measure by above-mentioned chromatographic condition sample introduction, record chromatogram, with peak area A for ordinate, concentration C is that horizontal ordinate carries out linear regression, result shows, each composition is good linear relation within the scope of respective concentration, the range of linearity, regression equation, related coefficient is in table 1.
Table 1 range of linearity, regression equation, related coefficient
Instrument precision is tested
Draw reference substance mixed solution (Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin concentration are 100ng/ml), measure by above-mentioned chromatographic condition sample introduction, repeat sample introduction 6 times, record peak area, calculate RSD.The RSD of the peak area of Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin is respectively: 0.34%, 4.22%, 0.98%, 2.28%, 0.45%, shows that instrument precision is good.
Repeatability
Get Rhizoma Gastrodiae extract (lot number 132100) and prepare need testing solution six parts by above-mentioned need testing solution preparation method is parallel, measure by above-mentioned chromatographic condition sample introduction, record chromatogram, the RSD of calculating Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin content is respectively 2.43%, 2.19%, 2.78%, 2.84%, show that this method repeatability is good.
Stability
Get Rhizoma Gastrodiae extract need testing solution (lot number 132100), at ambient temperature respectively at 0,3,6,9,12,24h measures by above-mentioned chromatographic condition sample introduction, record chromatogram, the RSD of calculating Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin content is respectively 2.42%, 4.70%, 2.51% and 4.96%, 1.78%.Result shows, need testing solution is stable in 24 hours.
Recovery test
Get totally 6 parts, Rhizoma Gastrodiae extract sample (lot number 132100), every part of 10mg, accurately weighed, put in 50ml measuring bottle, add Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin reference substance stock solution respectively in right amount, constant volume after dissolving with 8% methanol-water, measure by above-mentioned chromatographic condition sample introduction, record chromatogram, calculates the average recovery of Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin, the results are shown in Table 2.
Table 2 determination of recovery rates (n=6)
Sample determination
Get Rhizoma Gastrodiae extract (lot number 132100) and prepare need testing solution by above-mentioned need testing solution preparation method, measure by above-mentioned chromatographic condition sample introduction, record chromatogram, calculate the percentage composition of Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin, the results are shown in Table 3.
Table 3 Rhizoma Gastrodiae extract 132100 assay
The content detection of Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin in embodiment 3 Rhizoma Gastrodiae extract
Rhizoma Gastrodiae extract lot number is 132200.
Chromatographic condition: chromatographic column is AgilentZORBAXSB-C18 (3.0 × 100mm, 1.8 μm), and mobile phase is methyl alcohol (A)-0.1% formic acid solution (B), elution program: 0-5min, A linearly rises to 40%, 5-12min from 8%, and A maintains 40%; Column temperature: 40 DEG C; Flow velocity: 0.25ml/min; Excitation wavelength 215nm, emission wavelength 295nm; Sample size 1 μ l.
Prepared by reference substance solution
Get Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin reference substance respectively in right amount, accurately weighed, dissolve with 8% methanol-water and be diluted to the reference substance stock solution that concentration is 1mg/ml.
A certain amount of 5 kinds of reference substance stock solutions of accurate absorption, add 8% methanol-water and are diluted to the mixing reference substance solution that concentration is 100ng/ml
Prepared by test sample
Take Rhizoma Gastrodiae extract (lot number 132200) 10mg, accurately weighed, put in 50ml measuring bottle, add 8% methanol-water and dissolve and constant volume, shake up, both.
Sample determination
Get Rhizoma Gastrodiae extract (lot number 132200) and prepare need testing solution by above-mentioned need testing solution preparation method, measure by above-mentioned chromatographic condition sample introduction, record chromatogram, calculate the percentage composition of Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin, the results are shown in Table 4.
Table 4 Rhizoma Gastrodiae extract 132200 batches of assays
The content detection of Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin in embodiment 4 Rhizoma Gastrodiae
Chromatographic condition: chromatographic column is AgilentZORBAXSB-C18 (3.0 × 100mm, 1.8 μm), and mobile phase is methyl alcohol (A)-0.5% formic acid solution (B), elution program: 0-5min, A linearly rises to 40%, 5-12min from 8%, and A maintains 40%; Column temperature: 60 DEG C; Flow velocity: 0.35ml/min; Excitation wavelength 235nm, emission wavelength 335nm; Sample size 20 μ l.
Prepared by reference substance solution
Get Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin reference substance respectively in right amount, accurately weighed, dissolve with 8% methanol-water and be diluted to the reference substance stock solution that concentration is 1mg/ml.
A certain amount of 5 kinds of reference substance stock solutions of accurate absorption, add 8% methanol-water and are diluted to the mixing reference substance solution that concentration is 100ng/ml
Prepared by test sample
Take Rhizoma Gastrodiae powder (crossing No. three sieves) 2g, by the 70% alcohol-water refluxing extraction 3 times of 5 times, extract merges concentrated, with suitable quantity of water dispersion, and the extraction into ethyl acetate of 2 times of volumes 2 times, the water layer obtained merges concentrated, accurately weighed concentrate 10mg, puts in 50ml measuring bottle, adds 8% methanol-water and dissolves and constant volume, shake up, both.
Sample determination
Get Rhizoma Gastrodiae and prepare need testing solution by above-mentioned need testing solution preparation method, measure by above-mentioned chromatographic condition sample introduction, record chromatogram, calculates the percentage composition of Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin, the results are shown in Table 5.
Table 5 Rhizoma Gastrodiae assay
Claims (9)
1. the method for multiple phenols component content in Simultaneous Determination rhizoma Gastrodiae, is characterized in that: mainly comprise following three steps,
1) reference standards and the preparation for examination product to be tested solution: rhizoma Gastrodiae phenols reference standards and Rhizoma Gastrodiae extract to be measured being dissolved in respectively volumetric concentration is in the methyl alcohol of 0-8% or the aqueous solution of acetonitrile, preparation reference standards mixed solution and need testing solution to be measured;
2) with high performance liquid chromatography-fluorescence method for means carry out the quantitative test of reference standards mixed solution and need testing solution composition to be measured respectively;
Analyzed by the reference standards mixed solution of the rhizoma Gastrodiae phenolic compound to a series of different quality concentration, obtain the retention time of each rhizoma Gastrodiae phenolic compound in reference standards mixed solution and peak area corresponding to each rhizoma Gastrodiae phenolic compound different quality concentration;
By the analysis to need testing solution to be measured, obtain the retention time of rhizoma Gastrodiae phenolic compound and the peak area of correspondence in Rhizoma Gastrodiae extract to be measured;
3) content of phenols component in external standard method rhizoma Gastrodiae;
Analyzed by the reference standards mixed solution of the rhizoma Gastrodiae phenolic compound to a series of different quality concentration, using the known quality concentration of rhizoma Gastrodiae phenolic compound and corresponding peak area as transverse and longitudinal coordinate, set up each known rhizoma Gastrodiae phenolic compound typical curve;
With the retention time of rhizoma Gastrodiae phenolic compound each in reference standards mixed solution for benchmark, treating rhizoma Gastrodiae phenolic compound in observation nettle extract carries out qualitative, the peak area of rhizoma Gastrodiae phenolic compound in Rhizoma Gastrodiae extract to be measured after qualitative is substituted in its corresponding rhizoma Gastrodiae phenolic compound typical curve, it is carried out quantitatively.
2. method according to claim 1, it is characterized in that: step 1) in rhizoma Gastrodiae phenols reference standards comprise in Gastrodin, p-Hydroxybenzylalcohol, parahydroxyben-zaldehyde, parishinE, parishinG, parishinB, parishinC and parishin more than two kinds or three kinds, in reference standards mixed solution, the mass concentration of each material is 5-2500ng/ml.
3. method according to claim 2, is characterized in that: step 1) in rhizoma Gastrodiae phenols component mainly comprise in Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin more than two kinds or three kinds.
4. method according to claim 1, is characterized in that: step 1) in prepare Gastrodin, p-Hydroxybenzylalcohol, parishinB, parishinC and parishin reference substance mixed solution with 8% methanol-water for solvent and prepare Rhizoma Gastrodiae extract need testing solution.
5. method according to claim 1, is characterized in that: a series of different quality concentration refers to that rhizoma Gastrodiae phenolic compound in reference standards solution chooses more than 5, the concentration value of concentration interval at least one times between mass concentration 5-2500ng/ml.
6. method according to claim 1, is characterized in that:
Step 2) in: chromatographic condition is: C18 liquid-phase chromatographic column; Mobile phase is methyl alcohol or acetonitrile (A)-volume fraction 0.1 ~ 0.5% aqueous acid (B); Gradient program wash-out; Column temperature: 40 ~ 60 DEG C; Flow velocity: 0.25 ~ 0.35ml/min; Excitation wavelength 215nm ~ 235nm, emission wavelength 295 ~ 335nm; Sample size 1 ~ 20 μ l.
7. method according to claim 6, is characterized in that:
Step 2) in mobile phase be methyl alcohol-aqueous acid, sour preferable formic acid, its volume fraction is 0.5%;
Step 2) in column temperature be 50 DEG C, flow velocity is 0.35ml/min, and sample size is 1 μ l;
Step 2) in excitation wavelength be 225nm, emission wavelength is 310nm.
8. method according to claim 1, is characterized in that: the mass concentration 0.01-2mg/ml of Rhizoma Gastrodiae extract in need testing solution to be measured.
9. method according to claim 1, is characterized in that: Rhizoma Gastrodiae extract is the ethanol water extract of the water extract of rhizoma Gastrodiae, the ethanol extract of rhizoma Gastrodiae or rhizoma Gastrodiae.
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| CN108020615A (en) * | 2018-03-29 | 2018-05-11 | 宁波大学 | A kind of method of total gastrodia elata genin content in quantitative determination rhizoma Gastrodiae |
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| CN106732473B (en) * | 2016-11-14 | 2019-10-08 | 广西民族大学 | A method of the rosin based high molecular chromatographic column of esterification modification separates Gastrodin |
| CN107643345A (en) * | 2017-08-21 | 2018-01-30 | 王信 | A kind of method of quality control of Rhizoma Gastrodiae and its medicine materical crude slice and extract |
| CN108020615A (en) * | 2018-03-29 | 2018-05-11 | 宁波大学 | A kind of method of total gastrodia elata genin content in quantitative determination rhizoma Gastrodiae |
| CN111257437A (en) * | 2018-11-30 | 2020-06-09 | 华中科技大学 | Method for detecting various components in gastrodia elata, gastrodia elata extract and gastrodia elata-containing preparation |
| CN111257481A (en) * | 2018-11-30 | 2020-06-09 | 华中科技大学 | Method for establishing fingerprint of rhizoma gastrodiae medicinal material and fingerprint thereof |
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