CN104181241A - Determination method of forsythiaside A and forsythin content in fructus forsythiae leaves - Google Patents
Determination method of forsythiaside A and forsythin content in fructus forsythiae leaves Download PDFInfo
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- CN104181241A CN104181241A CN201310203252.XA CN201310203252A CN104181241A CN 104181241 A CN104181241 A CN 104181241A CN 201310203252 A CN201310203252 A CN 201310203252A CN 104181241 A CN104181241 A CN 104181241A
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- forsythiaside
- forsythin
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Abstract
The invention discloses a determination method of forsythiaside A and forsythin content in fructus forsythiae leaves, and the method comprises the following steps: the preparation of a to-be tested sample solution, the preparation of a reference solution, HPLC condition and standard curve formulation and result calculation. The method has the advantages of simple operation, strong specificity, good repeatability, good separating degree and reliable results.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical field, be specifically related to the content assaying method of composition.
Background technology
The capsule of weeping forsythia
forsythia suspensa(Thunb.) Vahl is Oleaceae plants Forsythia Vahl machaka.Its fruit is parts of generic medicinal plants, have clearing heat and detoxicating, dispersing swelling and dissipating binds, the effect of dispelling wind and heat from the body, have antipyretic significantly, antibacterial, antiviral effect.Forsythin and forsythiaside A have been listed the index components of version < < Chinese Pharmacopoeia > > assay in 2010 in as main effective constituent wherein.Have bibliographical information, some active constituent content in Folium Forsythia, higher than capsule of weeping forsythia fruit, has significantly anti-oxidant, the anti-ageing effect of waiting for a long time, and the conventional capsule of weeping forsythia tender leaf in some areas is made Forsythia suspense leaves tea and drunk, and has good health care and is worth.
Also there is at present the content method of measuring active component in Folium Forsythia, but poor repeatability, and effect is bad.
Summary of the invention
The object of this invention is to provide a kind of reproducible, result and measure accurately the assay method of forsythin and forsythiaside A content in Folium Forsythia.
The technical solution adopted in the present invention is:
An assay method for forsythiaside A and Determination of forsythin in Folium Forsythia, the method consists of following steps:
The preparation of A, test sample solution: precision takes Folium Forsythia and pulverizes sample 0.1-0.5g, adds 50-70% methyl alcohol 50ml, then weighs, and ultrasonic extraction 10-30min, weighs after letting cool, and methyl alcohol is supplied less loss weight, shakes up filtration, obtains test sample solution;
The preparation of B, reference substance solution: precision takes forsythiaside A, forsythin reference substance, is mixed with respectively forsythiaside A, the forsythin reference substance solution of variable concentrations with 50-70% methyl alcohol;
C, HPLC chromatographic condition: chromatographic column C
18post, mobile phase is methyl alcohol-0.1% phosphoric acid solution, and flow velocity is 1.0mL/min, and detection wavelength is 215-280nm, and column temperature is 30 ℃;
The formulation of D, typical curve and result of calculation: the forsythiaside A of variable concentrations, forsythin reference substance solution 6-10 μ l are analyzed under step C chromatographic condition, take peak area integrated value as ordinate, sample size is horizontal ordinate, draw the typical curve of forsythiaside A, forsythin, test sample solution is analyzed under step C chromatographic condition, by the peak area integrated value substitution typical curve of forsythiaside A, forsythin, obtain respectively the content of forsythiaside A, forsythin.
As preferably, in steps A, with 0.22 μ m filter membrane, filter.
As preferably, in step C, chromatographic column specification is 5 μ m, 4.6 * 150mm.
As preferably, in step C, the ratio of methyl alcohol-0.1% phosphoric acid solution is 27-37:73-63.
Below the degree of accuracy of the inventive method, the testing result of precision.
instrument and reagent
Waters high performance liquid chromatograph, comprises 2695 Separations Module, 2998 Photodiode Array Detector, workstation: Empower 2 Software Build 2154; Ultrapure water (Milli-Q Advantage A10 ultrapure water system); Methyl alcohol (Fisher, chromatographically pure); Other reagent are pure for analyzing; Forsythiaside A reference substance (lot number is 111810-201102, and for assay, content is in 93.2%) is from National Institute for Food and Drugs Control; Forsythin reference substance (lot number is 110821-200610) is from Nat'l Pharmaceutical & Biological Products Control Institute; Folium Forsythia is provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd, and lot number is 20120501,20120901,20120902,20121101.
methodological study
Specificity test: under above-mentioned chromatographic condition, draw respectively forsythiaside A reference substance solution, forsythin reference substance solution, need testing solution and blank solution and detect.Result feminine gender is noiseless, and forsythiaside A peak, forsythin peak are separated with other peaks good.
Linear relationship is investigated: accurate absorption concentration is 29.3170 μ g/ml respectively, 58.6340 μ g/ml, 73.2925 μ g/ml, 146.5850 μ g/ml, 219.8774 μ g/ml, each 10 μ l of the forsythiaside A reference substance solution of 366.4624 μ g/ml, with concentration be 33.3760 μ g/ml, 66.7520 μ g/ml, 83.4400 μ g/ml, 166.8800 μ g/ml, 250.3200 μ g/ml, each 10 μ l of the forsythin reference substance solution of 417.2000 μ g/ml, inject respectively high performance liquid chromatograph, by above-mentioned chromatographic condition, measure peak area, take peak area integrated value as ordinate, the sample size of forsythiaside A and forsythin (ng) is horizontal ordinate, drawing standard curve: calculate regression equation: Y
forsythosidea
=1281.3706X+4256.8182 r=0.999980, Y
forsythin=2034.5969X-10608.2670 r=0.999991, result shows that forsythiaside A is within the scope of 293.170～3664.624ng, forsythin sample size within the scope of 333.760～4172.000ng, the linear relationship that tool is good.
Precision test: get respectively forsythiaside A reference substance solution and forsythin reference substance solution (forsythiaside A 73.2925 μ g/ml, forsythin 83.4400 μ g/ml), continuous sample introduction 6 times, each 10 μ l, the RSD of result forsythiaside A and forsythin peak area is respectively 0.47%, 1.14%, and illustration method precision is good.
Stability test: the accurate need testing solution 10 μ l that draw embodiment 1, room temperature is placed, and respectively at 0,4,8,12,16,24,40h sample introduction measures, the RSD of result forsythiaside A and forsythin peak area is respectively 2.12%, 0.91%.Show that need testing solution is stable in 40h.
Replica test: get the sample in embodiment 1, get respectively 0.16g, 0.20g, tri-sample sizes of 0.24g, 3 parts of each sample sizes, accurately weighed, according to the preparation method of need testing solution, prepare need testing solution, by chromatographic condition sample introduction, measure, the average quality mark (n=9) that result records forsythiaside A is 20.8601 mg/g, the average quality mark (n=9) of forsythin is 19.1787 mg/g, and RSD% is respectively 1.54%, 1.01%, and illustration method is reproducible.
Average recovery test: get 9 parts, sample in embodiment 1, every part of 0.1g, accurately weighed, the forsythiaside A and the forsythin reference substance 70% methanol solution 50ml that add respectively high, medium and low concentration, prepare need testing solution, and sample introduction is measured, calculate recovery rate, the results are shown in Table 1.
Table 1 determination of recovery rates result (n=9)
3 sample detection
Get different batches Folium Forsythia powder, according to the preparation method of need testing solution, prepare need testing solution, by above-mentioned chromatographic condition sample introduction, measure, record chromatogram, the amount with forsythiaside A and forsythin in external standard method calculated by peak area sample, the results are shown in Table 2.
Table 2 sample determination result (n=3)
Owing to having adopted technique scheme, the technical progress that the present invention obtains is:
The inventive method is simple to operate, and specificity is strong, reproducible, and degree of separation is good, reliable results.
Embodiment
Below in conjunction with embodiment, the present invention is described in further details:
Sample preparation: get Folium Forsythia, dry, control below moisture to 15%, pulverize, obtain.
Embodiment 1
An assay method for forsythiaside A and Determination of forsythin in Folium Forsythia, consists of following steps:
The preparation of A, test sample solution: precision takes Folium Forsythia and pulverizes sample 0.5g, adds 50% methyl alcohol 50ml, then weighs, and ultrasonic extraction 10min, weighs after letting cool, and methyl alcohol is supplied less loss weight, shakes up, and with 0.22 μ m membrane filtration, obtains test sample solution;
B, the preparation of reference substance solution: precision takes forsythiaside A, forsythin reference substance, with 50% methyl alcohol, be mixed with respectively the forsythiaside A of variable concentrations, forsythin reference substance solution, the reference substance solution concentration of forsythiaside A is respectively 29.3170 μ g/ml, 58.6340 μ g/ml, 73.2925 μ g/ml, 146.5850 μ g/ml, 219.8774 μ g/ml, 366.4624 μ g/ml, the reference substance solution concentration of forsythin is respectively 33.3760 μ g/ml, 66.7520 μ g/ml, 83.4400 μ g/ml, 166.8800 μ g/ml, 250.3200 μ g/ml, 417.2000 μ g/ml,
C, HPLC chromatographic condition: chromatographic column Agilent ZORBAX SB-C
18, specification is 5 μ m, 4.6 * 150mm, and mobile phase is methyl alcohol-0.1% phosphoric acid solution, and the ratio of methyl alcohol-0.1% phosphoric acid solution is 32:68, and flow velocity is 1.0mL/min, and detection wavelength is 227nm, column temperature is 30 ℃;
The formulation of D, typical curve and result of calculation: the forsythiaside A of variable concentrations, each 6 μ l of forsythin reference substance solution are analyzed under step C chromatographic condition, take peak area integrated value as ordinate, sample size is horizontal ordinate, draw the typical curve of forsythiaside A, forsythin, test sample solution is analyzed under step C chromatographic condition, by the peak area integrated value substitution typical curve of forsythiaside A, forsythin, obtain respectively the content of forsythiaside A, forsythin, the content of forsythiaside A is 27.63mg, and Determination of forsythin is 16mg.
Embodiment 2
In a kind of Folium Forsythia, the assay method of forsythiaside A and Determination of forsythin is as follows:
The preparation of A, test sample solution: precision takes Folium Forsythia and pulverizes sample 0.1g, adds 70% methyl alcohol 50ml, then weighs, and ultrasonic extraction 20min, weighs after letting cool, and methyl alcohol is supplied less loss weight, shakes up, and with 0.22 μ m membrane filtration, obtains test sample solution;
B, the preparation of reference substance solution: precision takes forsythiaside A, forsythin reference substance, with 70% methyl alcohol, be mixed with respectively the forsythiaside A of variable concentrations, forsythin reference substance solution, the reference substance solution concentration of forsythiaside A is respectively 29.3170 μ g/ml, 58.6340 μ g/ml, 73.2925 μ g/ml, 146.5850 μ g/ml, 219.8774 μ g/ml, 366.4624 μ g/ml, the reference substance solution concentration of forsythin is respectively 33.3760 μ g/ml, 66.7520 μ g/ml, 83.4400 μ g/ml, 166.8800 μ g/ml, 250.3200 μ g/ml, 417.2000 μ g/ml,
C, HPLC chromatographic condition: chromatographic column Agilent ZORBAX SB-C
18, specification is 5 μ m, 4.6 * 150mm, and mobile phase is methyl alcohol-0.1% phosphoric acid solution, and the ratio of methyl alcohol-0.1% phosphoric acid solution is 27:73, and flow velocity is 1.0mL/min, and detection wavelength is 215nm, column temperature is 30 ℃;
The formulation of D, typical curve and result of calculation: the forsythiaside A of variable concentrations, each 10 μ l of forsythin reference substance solution are analyzed under step C chromatographic condition, take peak area integrated value as ordinate, sample size is horizontal ordinate, draw the typical curve of forsythiaside A, forsythin, test sample solution is analyzed under step C chromatographic condition, by the peak area integrated value substitution typical curve of forsythiaside A, forsythin, obtain respectively the content of forsythiaside A, forsythin, the content of forsythiaside A is 20.63mg, and Determination of forsythin is 10mg.
Precision test: the RSD of forsythiaside A and forsythin peak area is respectively 0.45%, 1.12%, and illustration method precision is good.
Stability test: the RSD of forsythiaside A and forsythin peak area is respectively 2.10%, 0.92%.Show that need testing solution is stable in 40h.
Replica test: the average quality mark (n=9) of forsythiaside A is 20.8701 mg/g, the average quality mark (n=9) of forsythin is 19.1887 mg/g, and RSD% is respectively 1.52%, 1.03%, and illustration method is reproducible.
Average recovery test: the results are shown in Table 3.
Table 3 determination of recovery rates result (n=9)
Embodiment 3
In a kind of Folium Forsythia, the assay method of forsythiaside A and Determination of forsythin is as follows:
The preparation of A, test sample solution: precision takes Folium Forsythia and pulverizes sample 0.2g, adds 60% methyl alcohol 50ml, then weighs, and ultrasonic extraction 30min, weighs after letting cool, and methyl alcohol is supplied less loss weight, shakes up, and with 0.22 μ m membrane filtration, obtains test sample solution;
B, the preparation of reference substance solution: precision takes forsythiaside A, forsythin reference substance, with 70% methyl alcohol, be mixed with respectively the forsythiaside A of variable concentrations, forsythin reference substance solution, the reference substance solution concentration of forsythiaside A is respectively 29.3170 μ g/ml, 58.6340 μ g/ml, 73.2925 μ g/ml, 146.5850 μ g/ml, 219.8774 μ g/ml, 366.4624 μ g/ml, the reference substance solution concentration of forsythin is respectively 33.3760 μ g/ml, 66.7520 μ g/ml, 83.4400 μ g/ml, 166.8800 μ g/ml, 250.3200 μ g/ml, 417.2000 μ g/ml,
C, HPLC chromatographic condition: chromatographic column Agilent ZORBAX SB-C
18, specification is 5 μ m, 4.6 * 150mm, and mobile phase is methyl alcohol-0.1% phosphoric acid solution, and the ratio of methyl alcohol-0.1% phosphoric acid solution is 37:63, and flow velocity is 1.0mL/min, and detection wavelength is 280nm, column temperature is 30 ℃;
The formulation of D, typical curve and result of calculation: the forsythiaside A of variable concentrations, each 8 μ l of forsythin reference substance solution are analyzed under step C chromatographic condition, take peak area integrated value as ordinate, sample size is horizontal ordinate, draw the typical curve of forsythiaside A, forsythin, test sample solution is analyzed under step C chromatographic condition, by the peak area integrated value substitution typical curve of forsythiaside A, forsythin, obtain respectively the content of forsythiaside A, forsythin, the content of forsythiaside A is 17.66mg, and Determination of forsythin is 10.56mg.
Precision test: the RSD of forsythiaside A and forsythin peak area is respectively 0.45%, 1.09%, and illustration method precision is good.
Stability test: the RSD of forsythiaside A and forsythin peak area is respectively 2.02%, 0.92%.Show that need testing solution is stable in 40h.
Replica test: the average quality mark (n=9) of forsythiaside A is 20.8602mg/g, the average quality mark (n=9) of forsythin is 19.1788 mg/g, and RSD% is respectively 1.53%, 1.03%, and illustration method is reproducible.
Average recovery test: the results are shown in Table 4.
Table 4 determination of recovery rates result (n=9)
Claims (4)
1. an assay method for forsythiaside A and Determination of forsythin in Folium Forsythia, is characterized in that the method consists of following steps:
The preparation of A, test sample solution: precision takes Folium Forsythia and pulverizes sample 0.1-0.5g, adds 50-70% methyl alcohol 50ml, then weighs, and ultrasonic extraction 10-30min, weighs after letting cool, and methyl alcohol is supplied less loss weight, shakes up filtration, obtains test sample solution;
The preparation of B, reference substance solution: precision takes forsythiaside A, forsythin reference substance, is mixed with respectively forsythiaside A, the forsythin reference substance solution of variable concentrations with 50-70% methyl alcohol;
C, HPLC chromatographic condition: chromatographic column C
18post, mobile phase is methyl alcohol-0.1% phosphoric acid solution, and flow velocity is 1.0mL/min, and detection wavelength is 215-280nm, and column temperature is 30 ℃;
The formulation of D, typical curve and result of calculation: the forsythiaside A of variable concentrations, forsythin reference substance solution 6-10 μ l are analyzed under step C chromatographic condition, take peak area integrated value as ordinate, sample size is horizontal ordinate, draw the typical curve of forsythiaside A, forsythin, test sample solution is analyzed under step C chromatographic condition, by the peak area integrated value substitution typical curve of forsythiaside A, forsythin, obtain respectively the content of forsythiaside A, forsythin.
2. assay method according to claim 1, is characterized in that with 0.22 μ m filter membrane, filtering in steps A.
3. assay method according to claim 1, is characterized in that in step C, chromatographic column specification is 5 μ m, 4.6 * 150mm.
4. assay method according to claim 1, the ratio that it is characterized in that methyl alcohol-0.1% phosphoric acid solution in step C is 27-37:73-63.
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| CN112946113A (en) * | 2021-02-01 | 2021-06-11 | 湖北医药学院 | Method for identifying genuine medicinal material Shiweir fructus forsythiae basal source |
| CN112946113B (en) * | 2021-02-01 | 2022-09-30 | 湖北医药学院 | Method for identifying genuine medicinal material Shiweir fructus forsythiae basal source |
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