CN103713067B - Ultra-high performance liquid chromatography method for determining content of rheum lhasaense - Google Patents
Ultra-high performance liquid chromatography method for determining content of rheum lhasaense Download PDFInfo
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Abstract
The invention relates to the field of medicament analysis, and discloses an ultra-high performance liquid chromatography method for determining the content of rheum lhasaense. The method comprises the steps of: by taking acetonitrile-0.1% phosphoric acid water solution as a mobile phase, gradiently eluting rheum lhasaense-carried sample solution to enter an ultra-high performance liquid chromatograph; carrying out gradient elution by taking the acetonitrile-0.1% phosphoric acid water solution as a mobile phase; setting a standard sample, determining the response values of deoxyrhaponticin peak, triptophenolide peak, and polydatin peak in the rheum lhasaense sample by using an ultraviolet detector so as to respectively correspond to sample sizes, and calculating the content of ingredients in the rheum lhasaense sample through comparing the size of the response values with a standard sample. According to the method, the analysis and detection time of the sample can be shortened, the method has the characteristics of being good in separation effect, accurate to determine, high in sensitivity, strong in specificity, simple and convenient for an analysis method and the like, is also capable of saving the usage amount of a solvent, thus having great application prospects.
Description
Technical field
The present invention relates to Pharmaceutical Analysis field, particularly a kind of Ultra Performance Liquid Chromatography method measuring Multiple components content in Lhasa rhubarb.
Background technology
Lhasa rhubarb is polygonaceae plant, mainly originates in the ground such as Tibet, Sichuan, Yunnan, dries be used as medicine with rhizome.At present, by identifying in physicochemical constant and spectral analysis containing multiple talan constituents in Lhasa rhubarb, wherein, content has been Rhapontin, deoxy-, Quzhazhigan and polygonin compared with the composition of horn of plenty.
Rhapontin, deoxy-is 5-hydroxyl-4 '-methoxyl Stilbene-3-O-glucopyranoside, chemical constitution is as follows:
Quzhazhigan is compound (E)-1-(3,5-dihydroxyphenyl)-2-(3-hydroxyl-4-O-β-D-glucopyranose phenyl) ethene, or be called 3,5,3 ', 4 '-tetrahydroxy Stilbene-3 '-O-beta-glucosidase is (also known as 3,5,4'-trihydroxy-Stilbene-3'-O-glucoside).Chemical structural formula is as follows:
Polygonin has another name called polydatin, and structural formula is:
At present, the method generally used in medicine and food analysis is high performance liquid chromatography.Efficient liquid phase chromatographic analysis cost is high, and use various filled column, capacity is little, and analyze biomacromolecule and inorganic ions difficulty, mobile phase consumption is large, and instrument price and daily servicing expense expensive, analysis time is long.
Summary of the invention
Goal of the invention of the present invention is to provide a kind of method of Contents of Main Components of the Lhasa rhubarb of Accurate Determining at short notice.
The method of mensuration Lhasa rhubarb provided by the invention three kinds of component contents, enters Ultra Performance Liquid Chromatography instrument by acetonitrile-0.1% phosphate aqueous solution (10: 90) for eluent gradient wash-out carries Lhasa rhubarb sample solution; Be that acetonitrile-0.1% phosphate aqueous solution (10: 90) carries out gradient elution with mobile phase;
Setting standard specimen, the response at Rhapontin, deoxy-peak, Quzhazhigan peak, polygonin peak in Lhasa rhubarb sample is measured by UV-detector, its sample size corresponding respectively, calculates the content of each component of Lhasa rhubarb sample by the size of its response and the comparison of standard specimen.
As preferably, the chromatographic column of described Ultra Performance Liquid Chromatography instrument is C18 post, particle diameter 1.7 μm, column length 50mm.
More preferably, described UV-detector determined wavelength 319nm, column temperature 30 DEG C, flow velocity 0.61ml/min, sampling volume 0.4 μ l.
More preferably, the concrete gradient of described gradient elution is as follows:
Principle of work of the present invention is that the extract of Lhasa rhubarb medicinal material injects Ultra Performance Liquid Chromatography instrument, chromatographic column is entered for eluent gradient wash-out carries extract by acetonitrile-0.1% phosphate aqueous solution (10: 90), with the chromatographic column of ultrahigh pressure liquid phase, principal ingredient Rhapontin, deoxy-peak, Quzhazhigan peak, polygonin peak are separated with other chemical compositions, make it arrive UV-detector successively and be identified; Detecting device measures the response at Rhapontin, deoxy-peak, Quzhazhigan peak, polygonin peak in Lhasa rhubarb sample, and its sample size corresponding is how many respectively, by the size of its response and the content relatively calculating each component in Lhasa rhubarb sample of standard specimen.
In the present invention UPLC mensuration Lhasa rhubarb, the more traditional high performance liquid chromatography of efficient liquid-phase chromatography method of Contents of Main Components shortens the time that sample analysis detects, there is good separating effect, measure accurately, highly sensitive, the features such as specificity is strong, analytical approach is easy, also can save the consumption of solvent; The method of the invention can be used for the change accurately grasping effective constituent in the processes such as breed breeding, cultivating and growing optimization, extraction processing, has direct using value.
Accompanying drawing explanation
Fig. 1 is the uv absorption spectra of Quzhazhigan in methyl alcohol;
Fig. 2 is the uv absorption spectra of polygonin in methyl alcohol;
Fig. 3 is the uv absorption spectra of Rhapontin, deoxy-in methyl alcohol;
Fig. 4 is the chromatogram that common HPLC measures principal ingredient in Lhasa rhubarb, 1-Quzhazhigan, 2-polygonin, 3-Rhapontin, deoxy-;
Fig. 5 is the chromatogram that UPLC measures each standard specimen; 5-1-Quzhazhigan, 5-2-polygonin, 5-3-Rhapontin, deoxy-;
Fig. 6 is that UPLC measures Lhasa rhubarb medicinal material principal ingredient chromatogram, 1-Quzhazhigan, 2-polygonin, 3-Rhapontin, deoxy-.
Embodiment
The invention discloses a kind of Ultra Performance Liquid Chromatography method measuring Lhasa rhubarb component content, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail, but do not form to the claims in the present invention protect to obtain scope restriction.
Embodiment 1:
The extract of Lhasa rhubarb medicinal material is injected Ultra Performance Liquid Chromatography instrument, chromatographic column is entered for eluent gradient wash-out carries the composition such as Rhapontin, deoxy-, Quzhazhigan by acetonitrile-0.1% phosphate aqueous solution (10: 90), with the chromatographic column of ultrahigh pressure liquid phase, Rhapontin, deoxy-is separated with other chemical compositions, makes it arrive UV-detector successively and be identified; Detecting device measures the response at Rhapontin, deoxy-peak, Quzhazhigan peak, polygonin peak in Lhasa rhubarb sample, and its sample size corresponding is how many respectively, by the size of its response and the content relatively calculating each component in Lhasa rhubarb sample of standard specimen.
1, determined wavelength is determined
Get after Rhapontin, deoxy-, Quzhazhigan and polygonin standard specimen dissolve with methyl alcohol respectively and measure ultraviolet spectrum, all there is ultraviolet absorption curve, and the maximum absorption wavelength of three kinds of compositions is all between 318.5nm ~ 319.6nm, therefore determined wavelength selects 319nm.Three kinds of compositions uv absorption spectra in methyl alcohol as Figure 1-3.
2, the contrast of common high performance liquid chromatography and Ultra Performance Liquid Chromatography instrument
Under the above-mentioned condition determined, Quzhazhigan absorption peak retention time about 1.3 minutes, polygonin absorption peak retention time about 1.5 minutes, Rhapontin, deoxy-retention time about 2.59 minutes; In Lhasa rhubarb sample chromatogram, each one-tenth swarming all can flow out chromatographic column in 4 minutes.Calculate the theoretical number of plates with Quzhazhigan standard specimen absorption peak and be greater than 15000, in Lhasa rhubarb medicinal material sample determination collection of illustrative plates, Quzhazhigan absorption peak theoretical cam curve is greater than 10000, Quzhazhigan and polygonin peak degree of separation are greater than 1.5, meet the requirement of HPLC assay method, HPLC measures the chromatogram of principal ingredient in Lhasa rhubarb as shown in Figure 4.
Embodiment 2:
Pulverize as meal after getting Lhasa rhubarb medicinal material drying, precision takes about 0.1g, puts in 150ml tool plug triangular flask, accurately add 75% methyl alcohol 50ml, weigh, ultrasonic 30 minutes, supply the weight of less loss with 75% methyl alcohol, with 0.22 μm of membrane filtration, be need testing solution.
Condition determination: the C-18 chromatographic column selecting ultrahigh pressure liquid phase; With acetonitrile-0.1% phosphoric acid solution (10:90) for mobile phase carries out gradient elution, flow velocity 0.61ml/min; Column temperature 30 DEG C; Determined wavelength 319nm; Sampling volume 0.4 μ l.Measurement result is as table 1 ~ 3, and UPLC measures the chromatogram of each standard specimen as shown in Figure 5, and UPLC measures Lhasa rhubarb medicinal material principal ingredient chromatogram as shown in Figure 6.
Table 1: the content of Quzhazhigan in different batches Lhasa rhubarb medicinal material
Table 2: the content of polygonin in different batches Lhasa rhubarb medicinal material
Table 3: the content of Rhapontin, deoxy-in different batches Lhasa rhubarb medicinal material
Embodiment 3: methodology is examined or check
(1) reference substance and sample preparation
The preparation of 1.1 Quzhazhigan reference substance solution
Precision takes Quzhazhigan reference substance 0.0100g, puts in 50mL measuring bottle, adds ethanol and dissolves and be diluted to scale, shake up, and obtains (containing Quzhazhigan 0.2mg in every lmL).Precision measures reference substance solution 0.50ml, 1.00ml, 1.50ml, 2.00ml, 2.50ml, 3.50ml, put in 10mL measuring bottle respectively, add ethanol to scale, shake up, obtain the reference substance solution that concentration is 0.01mg/ml, 0.02mg/ml, 0.03mg/ml, 0.04mg/ml, 0.05mg/ml, 0.07mg/ml.
The preparation of 1.2 polygonin reference substance solution
Precision takes polygonin reference substance 0.0100g, puts in 50mL measuring bottle, adds ethanol and dissolves and be diluted to scale, shake up, and obtains (containing polygonin 0.2mg in every lmL).Precision measures reference substance solution 0.10ml, 0.25ml, 0.50ml, 0.75ml, 1.00ml, 1.25ml, put in 10mL measuring bottle respectively, add ethanol to scale, shake up, obtain the reference substance solution that concentration is 0.002mg/ml, 0.005mg/ml, 0.01mg/ml, 0.015mg/ml, 0.02mg/ml, 0.025mg/ml.
The preparation of 1.3 methyl polygonin reference substance solution
Precision takes methyl polygonin reference substance 0.0100g, puts in 50mL measuring bottle, adds ethanol and dissolves and be diluted to scale, shake up, and obtains (containing methyl polygonin 0.2mg in every lmL).Precision measures reference substance solution 0.25ml, 0.50ml, 0.75ml, 1.00ml, 1.50ml, 1.75ml, 2.00ml, put in 10mL measuring bottle respectively, add ethanol to scale, shake up, obtain the reference substance solution that concentration is 0.005mg/ml, 0.01mg/ml, 0.015mg/ml, 0.02mg/ml, 0.03mg/ml, 0.035mg/ml, 0.04mg/ml.
The preparation of 1.4 sample solutions
Take Lhasa rhubarb powder and be about 0.1g, accurately weighed, empirically design is extracted, and filter after being cooled to room temperature, filtrate is put in the volumetric flask of 100mL, and solubilizer is settled to scale, shakes up, and 0.22 μm of filter membrane filters, and discards just filtrate, gets subsequent filtrate, to obtain final product.
(2) sample introduction precision
Get Quzhazhigan, polygonin and methyl polygonin reference substance solution 0.4 μ L, repeat sample introduction 6 times, record the situation of change of peak area and the retention time measured for each time, and calculate relative standard deviation, carry out precision investigation, the results are shown in Table 4 ~ 6.
Table 4: Quzhazhigan peak area and retention time precision result
Table 5: polygonin peak area and retention time precision result
Table 6: Rhapontin, deoxy-peak area and retention time precision result
(3) repeatability
Get each about 0.1g of Lhasa rhubarb medicinal material coarse powder 6 parts, accurately weighed, measure by embodiment 1 method respectively, measure content and the retention time of Quzhazhigan, polygonin and methyl polygonin, calculate its relative standard deviation, carry out repeatability and investigate, the results are shown in Table 7 ~ 9.
Table 7: the method repeatability result of Quzhazhigan
Table 8: the method repeatability result of polygonin
Table 9: the method repeatability result of Rhapontin, deoxy-
(4) stability of solution
Get Lhasa rhubarb test sample and Quzhazhigan, polygonin and Rhapontin, deoxy-reference substance are appropriate, make need testing solution and reference substance solution in accordance with the law, measured at 0,6,12,24,36,48 hour respectively, record chromatogram, investigate the stability of reference substance and need testing solution, the results are shown in Table 10 ~ 12.
Table 10: Quzhazhigan peak area, retention time stability result
Table 11: polygonin peak area, retention time stability result
Table 12: Rhapontin, deoxy-peak area, retention time stability result
(5) linear
Respectively by the Quzhazhigan reference substance of variable concentrations, polygonin reference substance, Rhapontin, deoxy-reference substance solution continuous sample introduction successively, measure according to by embodiment 1 method, with the concentration of reference substance solution for horizontal ordinate (X), peak area value is ordinate (Y) drawing standard curve, calculates regression equation (table 13 ~ 15).
Table 13: the linear relationship of Quzhazhigan peak area and concentration
Its regression equation is: y=2.8223x-0.0056r=0.9999; The Quzhazhigan concentration range of linearity: 0.01 ~ 0.07mgmL
-1.
Table 14: the linear relationship of polygonin peak area and concentration
Its regression equation is: y=2.0942x-0.0011r=0.9999; The polygonin concentration range of linearity: 0.002 ~ 0.025mgmL
-1.
Table 15: the linear relationship of Rhapontin, deoxy-peak area and concentration
Its regression equation is: y=2.5239x+0.0007r=0.9998; The Rhapontin, deoxy-concentration range of linearity: 0.005 ~ 0.04mgmL
-1.
(6) recovery
Get Lhasa rhubarb medicinal material coarse powder and be about 0.05g, accurately weighed, accurate methyl polygonin solution 2.7mL, 1.6mL and 2.1mL adding the Quzhazhigan solution of 0.0502mg/mL, the polygonin solution of 0.0151mg/mL and 0.0302mg/mL respectively; 4.5mL, 2.7mL and 3.5mL; 6.4mL, 3.8mL and 5.0mL, measure according to by embodiment 1 method respectively, calculates the recovery and get final product, the results are shown in Table 16 ~ 18.
Table 16: the average recovery test findings of Quzhazhigan
Table 17: the average recovery test findings of polygonin
Table 18: the average recovery test findings of Rhapontin, deoxy-
Result shows: the mean value of Quzhazhigan average recovery is 96.72%, RSD is 0.50%, the mean value of polygonin average recovery is 98.17%, RSD is 0.65%, the mean value of methyl polygonin average recovery is 97.92%, RSD is 0.81%, this shows the content measuring Quzhazhigan in Lhasa rhubarb medicinal material, polygonin and methyl polygonin by the method, result is accurate, and method is reliable.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. measure a method for Lhasa rhubarb component content, it is characterized in that:
Acetonitrile/0.1% phosphate aqueous solution being 10: 90 by volume ratio is that eluent gradient wash-out carries Lhasa rhubarb sample solution and enters Ultra Performance Liquid Chromatography instrument; To take mobile phase as volume ratio be 10: 90 acetonitrile/0.1% phosphate aqueous solution carry out gradient elution;
Setting standard specimen, the response at Rhapontin, deoxy-peak, Quzhazhigan peak, polygonin peak in Lhasa rhubarb sample is measured by UV-detector, its sample size corresponding respectively, calculates the content of each component of Lhasa rhubarb sample by the size of its response and the comparison of standard specimen; The chromatographic column of described Ultra Performance Liquid Chromatography instrument is C18 post, particle diameter 1.7 μm, column length 50mm, flow velocity 0.61ml/min; The concrete gradient of described gradient elution is as follows:
2. method according to claim 1, is characterized in that, described UV-detector determined wavelength 319nm, column temperature 30 DEG C, sampling volume 0.4 μ l.
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| CN104224813B (en) * | 2014-09-03 | 2017-04-19 | 昆药集团股份有限公司 | Pharmaceutical composition as well as preparation method and application thereof |
| CN106290645B (en) * | 2016-08-23 | 2019-04-16 | 昆药集团股份有限公司 | A kind of construction method and its standard finger-print of Lhasa rhubarb finger-print |
| CN110146614A (en) * | 2019-05-25 | 2019-08-20 | 青海省药品检验检测院 | The detection method of ponticin in a kind of compound rough gentian Sodium Bicarbonate Tablets |
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