CN102914609A - Method for simultaneously detecting contents of four active ingredients in herba erigerontis preparation - Google Patents
Method for simultaneously detecting contents of four active ingredients in herba erigerontis preparation Download PDFInfo
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Abstract
The invention relates to a method for simultaneously detecting contents of four active ingredients in a herba erigerontis preparation. Content determination of the four active ingredients is performed after preparation of reference substance solution and test product solution. The method comprises injecting the four active ingredients in a liquid chromatograph, determining, recording a chromatogram, and obtaining the contents of the four active ingredients by calculation according to peak areas in an external standard method. Octadecyl silane bonded silica gel serves as a filling agent of the liquid chromatograph, one or more of acetonitrile or methanol can serve as a mobile phase A, one or more of 0.05%-1.0% of formic acid or acetic acid or phosphoric acid can serve as a mobile phase B, and elution is performed according to the following gradients: time of 0-10min and A at a proportion of 7%-9%; time of 10-24min and A at a proportion of 8%-12%; time of 24-37min and A at a proportion of 9%-16%; time of 37-57min and A at a proportion of 10%-24%; time of 57-60min and A at a proportion of 24%-7%. The method is strong in specificity, high in precision, good in repeatability, high in accuracy and good in durability.
Description
Technical field
The present invention relates to a kind of fleabane preparation Content of Chlorogenic Acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, these four kinds of methods that active constituent content detects simultaneously of lamp-dish flower acetic.
Background technology
Erigeron breviscapus begins to be stated from " the southern regions of the Yunnan Province book on Chinese herbal medicine ", practises claiming fleabane flower, and because spending like oil lamp, root is gained the name like the root of Chinese wild ginger.Have another name called oil lamp chrysanthemum, root of Chinese wild ginger grass, the native root of Chinese wild ginger, be exposed to the sun, two sunflower, eastern chrysanthemum, push up grass (Yunnan), be the dry herb of feverfew Erigeron breviscapus (Erigeron breviscapus), main product in Yunnan, the provinces and regions such as Sichuan, Guizhou, Guangxi, Hunan, Tibet.Erigeron breviscapus nature and flavor " sweet, warm, little hardship, Gan Xin " have expelling cold and relieving exterior syndrome, dispel rheumatism, the effect of promoting blood circulation and removing blood stasis, clearing and activating the channels and collaterals, pain relieving.It is as treatment and prevent the disease such as cardiovascular to have the vegetable drug of special efficacy, and market outlook are wide, and economic worth is very high." Chinese pharmacopoeia version in 1977 was once recorded, and 2005 editions are recorded again.Fleabane preparation just begins for clinical from the seventies in last century.The erigeron breviscapus liquid preparation is the medicinal extract that contains phenolic acid class and flavone compound isoreactivity composition that extracts take fleabane flower as raw material, adds formulated mixture, oral liquid, syrup and the eye drops of auxiliary material.Mixture, oral liquid, syrup are hemiplegia caused to the match for the treatment of extravasated blood card cerebral infarction, rheumatalgia, coronary disease and angina pectoris, eyeground retinal vein obstruction, and the illnesss such as vasculitic skin disease have good curative effect.Eye drops is original to eye class curative effect of disease such as treatment cataract.
Oral liquid formulations take the erigeron breviscapus mixture as representative has now confirmed for treatment ICVD effect remarkable.The erigeron breviscapus liquid preparation is compared other fleabane flower oral formulations, there is mouthfeel suitable, dispersion degree is large, absorb soon, can bring into play more rapidly curative effect, the advantage such as take and easy to carry, topmost also have, what its raw material adopted is the extract of fleabane flower, keeps as much as possible two large main active, i.e. phenolic acid class and flavone compounds of erigeron breviscapus.This also is that the fleabane flower liquid preparation compares the place that simple oral formulations take Breviscapinun as raw material has superiority in curative effect.Flavonoids is take Breviscapinun as representative; the phenolic acid class comprises caffeic acid, chlorogenic acid, DCQ series etc.; exist in the time of two large constituents and cause that product has that more significant anti-inflammatory protects the liver, anti-oxidant, anticoagulation, expansion of cerebral vascular, reduction cerebral vascular resistance; increase cerebral blood flow (CBF), little obstacle, the anti-miocardial infarction followed of control, increase the multiple biologically actives such as hat arterial flow.
At present, the existing quality standard of erigeron breviscapus liquid preparation series of products is drafted all more coarse, for example the act.std of erigeron breviscapus mixture (standard No. YBZ00612004-2010Z): adopt spectrophotometry general flavone content, liquid-phase chromatography method to measure lamp-dish flower acetic (having another name called scutellarin) content, carry out the thin-layered chromatography discriminating with erigeron breviscapus control medicinal material and caffeic acid reference substance.The quality condition of this standard weight point control Flavonoid substances because thin-layered chromatography method itself only can qualitative detection, can't quantitatively detect, and therefore can't know the quality condition of understanding liposoluble ingredient, and method is weak, poor controllability.Important activity composition in the preparation such as chlorogenic acid, DCQ etc. do not have method to carry out quality control yet in addition, therefore, are necessary the control method of existing quality standard is further improved.
Summary of the invention
The purpose of this invention is to provide four kinds of methods that active constituent content detects simultaneously in a kind of erigeron breviscapus liquid preparation, can further know phenolic acid class and the important active constituent content situation of this two large class of flavonoids in the erigeron breviscapus liquid preparation, be beneficial to the quality control of product, guarantee the stability of Product Process, guarantee that the product clinical effectiveness of different batches is consistent.
The present invention is with chlorogenic acid, caffeic acid, 1, and 3-O-dicaffeoylquinic acid, lamp-dish flower acetic are index components, has set up the method that adopts these four kinds of compounds contents in the hplc simultaneous determination erigeron breviscapus liquid preparation.
Concrete detection method is as follows:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 2-60 μ g/ml, contain caffeic acid 3.1-93 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 1.2-36 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 10-300 μ g/ml, for subsequent use.
2) preparation of need testing solution: get 1 mL or 1g fleabane preparation in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: draw respectively each 10-20 μ L of reference substance solution and need testing solution, in the injection liquid chromatography, measure, the record chromatogram is pressed external standard method with calculated by peak area, and be get final product;
Wherein: the liquid chromatograph octadecylsilane chemically bonded silica is filling agent; In acetonitrile or the methyl alcohol one or more are as mobile phase A, and the aqueous solution of one or more in the formic acid of 0.05%-1.0% or acetic acid or the phosphoric acid is Mobile phase B, by following gradient elution:
Time 10～24min, ratio 8%～12%A;
Time 24～37min, ratio 9%～16%A;
Time 37～57min, ratio 10%～24%A;
Time 57～60min, ratio 24%～7%A;
Flow velocity per minute 0.5-2mL; Detect wavelength 327nm; Column temperature 30-40 ℃, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
The raw material medicinal extract that method of the present invention is applicable to these the three kinds of formulations itself of Herba Erigerontis oral liquid, syrup and eye drops in the fleabane preparation and extracts, and the sugar-free of oral liquid and mixture and have two kinds of specifications of sugar applicable too.
The present invention has the following advantages compared with the prior art: the act.std of erigeron breviscapus liquid preparation is all more coarse, mostly adopts liquid phase process to measure the content of lamp-dish flower acetic, differentiates with erigeron breviscapus control medicinal material and caffeic acid thin-layer chromatography.This standard is only controlled the quality of Flavonoid substances lamp-dish flower acetic, and thin layer method but can't be known the quality condition of understanding the phenolic acid class in the mixture.This method has been selected chlorogenic acid, caffeic acid, 1, and 3-O-dicaffeoylquinic acid, these four compositions of lamp-dish flower acetic adopt high performance liquid chromatography to measure simultaneously.Method is simple to operate, and is reliable and stable, can be used as the method for enterprises quality control, and replenishing of act.std guarantees the technology stability of product, guarantees that the product clinical effectiveness of different batches is consistent.The method specificity is strong, precision is high, good reproducibility, accuracy are high, durability good, be applicable to erigeron breviscapus liquid preparation Content of Chlorogenic Acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, these four component concentrations of lamp-dish flower acetic mensuration simultaneously.
Because source, the extracting method of the raw material of erigeron breviscapus mixture, oral liquid, syrup and eye drops are all consistent, although this paper only encloses the research process of mixture, but the methodology checking by actual each kind, can determine the raw material medicinal extract that method of the present invention is equally applicable to these three kinds of formulations itself of oral liquid, syrup and eye drops and extracts, and the sugar-free of oral liquid and mixture and have two kinds of specifications of sugar applicable too.
Description of drawings
Fig. 1 is solvent blank HPLC chromatogram.
Fig. 2 is for there being sugared type mixture negative sample HPLC chromatogram.
Fig. 3 is Sugarless type mixture negative sample HPLC chromatogram.
Fig. 4 is that four components are mixed contrast HPLC chromatogram.
Fig. 5 is for there being sugared type mixture sample HPLC chromatogram.
Fig. 6 is Sugarless type mixture sample HPLC chromatogram.
Embodiment
Instrument and reagent: Shimadzu 10A high performance liquid chromatograph, Weil-McLain dragon chromatographic work station, BSA224S-CW type electronic analytical balance (Sai Duolisi scientific instrument Beijing company limited), AS3120A supersonic wave cleaning machine;
The erigeron breviscapus mixture has sugared type, erigeron breviscapus mixture Sugarless type, Herba Erigerontis oral liquid to have sugared type, Herba Erigerontis oral liquid Sugarless type, erigeron breviscapus syrup, erigeron breviscapus eye drops to be provided by Kunming Zhenhua Pharmacy Co., Ltd.;
Chlorogenic acid (110753-200413, purity〉98%), caffeic acid (110885-200102, purity〉98%), 1,3-O-dicaffeoylquinic acid (111717-200501, purity〉98%) and lamp-dish flower acetic (have another name called scutellarin, 11842-201106, purity〉98%) reference substance all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
Experimental water is ultrapure water; Methyl alcohol, acetonitrile are chromatographically pure.
Embodiment 1:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 20 μ g/ml, contain caffeic acid 30 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 12 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 100 μ g/ml, for subsequent use;
2) preparation of need testing solution: get 1 mL erigeron breviscapus mixture (Sugarless type) in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, in the injection liquid chromatography, measures, and the record chromatogram is pressed external standard method with calculated by peak area, and be get final product; Wherein:
The liquid chromatograph octadecylsilane chemically bonded silica is filling agent; Take acetonitrile as mobile phase A, 0.1% aqueous formic acid is Mobile phase B, presses following gradient elution:
Time 10～24min, ratio 8%～9%A;
Time 24～37min, ratio 9%～10%A;
Time 37～57min, ratio 10%～24%A;
Time 57～60min, ratio 24%～7%A;
Flow velocity per minute 1.0mL; Detect wavelength 327nm; 30 ℃ of column temperatures, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
Result: record erigeron breviscapus mixture (Sugarless type) Content of Chlorogenic Acid 157.9 μ gmL
-1, caffeic acid 225.95 μ gmL
-1, 1,3-O-dicaffeoylquinic acid, 73.10 μ gmL
-1, lamp-dish flower acetic 1287.22 μ gmL
-1
Embodiment 2:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 20 μ g/ml, contain caffeic acid 18 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 10 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 100 μ g/ml, for subsequent use;
2) preparation of need testing solution: get 1 mL erigeron breviscapus mixture (sugared type is arranged) in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, in the injection liquid chromatography, measures, and the record chromatogram is pressed external standard method with calculated by peak area, and be get final product; Wherein:
The liquid chromatograph octadecylsilane chemically bonded silica is filling agent; Take acetonitrile-methyl alcohol (1:1) as mobile phase A, 0.1% aqueous formic acid is Mobile phase B, presses following gradient elution:
Time 10～24min, ratio 8%～10%A;
Time 24～37min, ratio 10%～10%A;
Time 37～57min, ratio 10%～24%A;
Time 57～60min, ratio 24%～12%A;
Flow velocity per minute 1.0mL; Detect wavelength 327nm; 30 ℃ of column temperatures, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
Result: record erigeron breviscapus mixture (sugared type is arranged) Content of Chlorogenic Acid 140 μ gmL
-1, caffeic acid 177.6 μ gmL
-1, 1,3-O-dicaffeoylquinic acid, 66.36 μ gmL
-1, lamp-dish flower acetic 1003.6 μ gmL
-1
Embodiment 3:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 12 μ g/ml, contain caffeic acid 15 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 6.0 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 50 μ g/ml, for subsequent use;
2) preparation of need testing solution: get 1 mL Herba Erigerontis oral liquid (Sugarless type) in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, in the injection liquid chromatography, measures, and the record chromatogram is pressed external standard method with calculated by peak area, and be get final product; Wherein:
The liquid chromatograph octadecylsilane chemically bonded silica is filling agent; Take methyl alcohol as mobile phase A, 1.0% acetic acid aqueous solution is Mobile phase B, presses following gradient elution:
Time 10～24min, ratio 8%～11%A;
Time 24～36min, ratio 11%～16%A;
Time 36～57min, ratio 16%～24%A;
Time 57～60min, ratio 24%～7%A;
Flow velocity per minute 2.0mL; Detect wavelength 327nm; 30 ℃ of column temperatures, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
Result: record Herba Erigerontis oral liquid (Sugarless type) Content of Chlorogenic Acid 140.2 μ gmL
-1, caffeic acid 177.0 μ gmL
-1, 1,3-O-dicaffeoylquinic acid, 66.30 μ gmL
-1, lamp-dish flower acetic 1003.5 μ gmL
-1
Embodiment 4:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 25 μ g/ml, contain caffeic acid 30 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 12 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 120 μ g/ml, for subsequent use;
2) preparation of need testing solution: get 1 mL Herba Erigerontis oral liquid (sugared type is arranged) in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: precision is drawn reference substance solution and each 20 μ L of need testing solution respectively, in the injection liquid chromatography, measures, and the record chromatogram is pressed external standard method with calculated by peak area, and be get final product; Wherein:
The liquid chromatograph octadecylsilane chemically bonded silica is filling agent; Take acetonitrile as mobile phase A, 0.05% aqueous formic acid is Mobile phase B, presses following gradient elution:
Time 10～24min, ratio 9%～12%A;
Time 24～37min, ratio 12%～16%A;
Time 37～57min, ratio 16%～24%A;
Time 57～60min, ratio 24%～7%A;
Flow velocity per minute 0.5mL; Detect wavelength 327nm; 40 ℃ of column temperatures, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
Result: record Herba Erigerontis oral liquid (sugared type is arranged) Content of Chlorogenic Acid 164.3 μ gmL
-1, caffeic acid 239.0 μ gmL
-1, 1,3-O-dicaffeoylquinic acid, 78.1 μ gmL
-1, lamp-dish flower acetic 1319.0 μ gmL
-1
Embodiment 5:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 60 μ g/ml, contain caffeic acid 93 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 36 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 300 μ g/ml, for subsequent use;
2) preparation of need testing solution: get 1mL erigeron breviscapus syrup in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, in the injection liquid chromatography, measures, and the record chromatogram is pressed external standard method with calculated by peak area, and be get final product; Wherein:
The liquid chromatograph octadecylsilane chemically bonded silica is filling agent; Take acetonitrile as mobile phase A, 0.1% aqueous formic acid is Mobile phase B, presses following gradient elution:
Time 10～24min, ratio 9%～10%A;
Time 24～37min, ratio 10%～14%A;
Time 37～57min, ratio 14%～24%A;
Time 57～60min, ratio 24%～10%A;
Flow velocity per minute 1.5mL; Detect wavelength 327nm; 35 ℃ of column temperatures, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
Result: record erigeron breviscapus syrup Content of Chlorogenic Acid 164.4 μ gmL
-1, caffeic acid 237.8 μ gmL
-1, 1,3-O-dicaffeoylquinic acid, 77.85 μ gmL
-1, lamp-dish flower acetic 1327.0 μ gmL
-1
Embodiment 6:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 2 μ g/ml, contain caffeic acid 3.1 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 1.2 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 10 μ g/ml, for subsequent use;
2) preparation of need testing solution: get 1 mL erigeron breviscapus eye drops in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, in the injection liquid chromatography, measures, and the record chromatogram is pressed external standard method with calculated by peak area, and be get final product; Wherein:
The liquid chromatograph octadecylsilane chemically bonded silica is filling agent; Take acetonitrile as mobile phase A, 0.1% phosphate aqueous solution is Mobile phase B, presses following gradient elution:
Time 10～24min, ratio 9%～12%A;
Time 24～37min, ratio 12%～16%A;
Time 37～57min, ratio 16%～24%A;
Time 57～60min, ratio 24%～8%A;
Flow velocity per minute 1.2mL; Detect wavelength 327nm; 35 ℃ of column temperatures, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
Result: record erigeron breviscapus eye drops Content of Chlorogenic Acid 155.2 μ gmL
-1, caffeic acid 190.4 μ gmL
-1, 1,3-O-dicaffeoylquinic acid, 79.6 μ gmL
-1, lamp-dish flower acetic 1120.32 μ gmL
-1
Embodiment 7:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 60 μ g/ml, contain caffeic acid 93 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 36 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 300 μ g/ml, for subsequent use;
2) preparation of need testing solution: get 1 mL erigeron breviscapus medicinal extract (proportion 1.1) in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, in the injection liquid chromatography, measures, and the record chromatogram is pressed external standard method with calculated by peak area, and be get final product; Wherein:
The liquid chromatograph octadecylsilane chemically bonded silica is filling agent; Take acetonitrile as mobile phase A, 0.08% acetic acid aqueous solution is Mobile phase B, presses following gradient elution:
Time 10～24min, ratio 8%～10%A;
Time 24～37min, ratio 10%～15%A;
Time 37～57min, ratio 15%～24%A;
Time 57～60min, ratio 24%～10%A;
Flow velocity per minute 1.0mL; Detect wavelength 327nm; 30 ℃ of column temperatures, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
Result: record erigeron breviscapus medicinal extract (proportion 1.1) Content of Chlorogenic Acid 320 μ gmL
-1, caffeic acid 500 μ gmL
-1, 1,3-O-dicaffeoylquinic acid, 160 μ gmL
-1, lamp-dish flower acetic 2650.0 μ gmL
-1
The method of detection of the present invention is verified by the side of following several respects.
Take the methodological study of the HPLC of erigeron breviscapus mixture as example:
1, method interference test: do not contain the mixture that erigeron breviscapus extracts medicinal extract by producing the prescription preparation, by step 2) is made negative sample solution; Get reference substance solution, each 10 μ L of need testing solution and negative sample solution, sample introduction is measured, record chromatogram, result: under above-mentioned chromatographic condition, solvent blank, have sugared type negative sample and Sugarless type negative sample all noiseless to measuring.
2, the investigation of linear relationship: it is an amount of that precision pipettes mixing reference substance stock solution, and it is 2,10,20,40 that preparation contains chlorogenic acid, 60 μ gmL
-1Containing caffeic acid is 3.1,15.5,31,62,93 μ gmL
-1Containing 1,3-O-dicaffeoylquinic acid is 1.2,6.0,12,24,36 μ gmL
-1Containing lamp-dish flower acetic is 10.0,50.0,100.0,200,300.0 μ gmL
-1The mixing reference substance solution, shake up, the mixing reference substance solution 10 μ L that draw respectively above-mentioned concentration gradient inject high performance liquid chromatograph.Respectively with reference substance concentration (μ gmL
-1) be horizontal ordinate, the peak area integrated value is ordinate, and the drawing standard curve line retrace of going forward side by side calculates, and gets chlorogenic acid, caffeic acid, 1, and typical curve regression equation and the range of linearity of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic the results are shown in Table 1.The result shows: in the range of linearity, linear relationship is good.
(n=6) investigated in 4 kinds of component lines sexual intercourse of table 1
| Compound | Regression equation | The range of linearity/μ g | r |
| Chlorogenic acid | Y=2.92×10-5X-0.709 | 0.020～0.600 | 0.9990 |
| Caffeic acid | Y=1.65×10-5X+0.669 | 0.031～0.930 | 0.9999 |
| 1,3-O-dicaffeoylquinic acid | Y=1.77×10-5X+0.463 | 0.012～0.360 | 0.9999 |
| Lamp-dish flower acetic | Y=3.08×10-5X-0.214 | 0.100～3.000 | 0.9995 |
3, precision test: get erigeron breviscapus mixture (Sugarless type, lot number 20120901), use above-mentioned need testing solution, precision measures 10 μ L and injects high performance liquid chromatograph, repeats sample introduction 6 times, the record peak area, and the result shows: chlorogenic acid RSD 1.13%; Caffeic acid RSD 1.11%; 1,3-O-dicaffeoylquinic acid RSD O.42%; Lamp-dish flower acetic RSD O.5%.
4, replica test: get erigeron breviscapus mixture (Sugarless type, lot number 20120901), with 6 parts of above-mentioned need testing solutions, precision measures 10 μ L and injects high performance liquid chromatograph, the record peak area, utilize working curve to calculate content, record each composition average quality concentration and be followed successively by chlorogenic acid 157.9 μ gmL
-1, caffeic acid 226.0 μ gmL
-1, 1,3-O-dicaffeoylquinic acid, 73.1 μ gmL
-1, lamp-dish flower acetic 1287.2 μ gmL
-1RSD is followed successively by 1.07%, 0.98%, and 1.71%, 1.52%.
5, stability test: get erigeron breviscapus mixture (Sugarless type, lot number 20120901), use above-mentioned need testing solution, room temperature is placed, and respectively at 0,1,2,4,6,8,12h, repeats sample introduction 6 times, the record peak area.The result shows: chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic are stable in 12h, and RSD is respectively 1.19%, 0.4%, and 1.01%, 1.17%.
6, recovery test: the erigeron breviscapus mixture 1.0mL that gets known content, place 10 mL measuring bottles, add respectively chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic storing solution are an amount of, use above-mentioned need testing solution, 6 parts of operation repetitives detect the content of each composition, calculate recovery rate the results are shown in Table 2.
The average recovery (n=6) of 4 kinds of compositions of table 2
7, system's serviceability test is investigated: use above-mentioned need testing solution, selecting different column temperatures, different brands chromatographic column, different experiments personnel and different liquid chromatographs is that 20120901 sample is measured to the Sugarless type lot number.
Table 3 serviceability test---column temperature is investigated
Table 4 serviceability test---chromatographic column is investigated (40 ℃)
Table 5 serviceability test---liquid chromatograph is investigated
Adopt DAD to carry out spectral scan, the blending ingredients reference substance locates that at 327,335 nm large absorption peak is all arranged most, but under the 335 nm conditions, the peak shape of chromatographic peak is relatively poor.Under the 327 nm conditions, can satisfy basic separation requirement.The theoretical cam curve of four components is higher than 5000.Consider, selecting to detect wavelength is 327 nm.
Because source, the extracting method of the raw material of erigeron breviscapus mixture, oral liquid, syrup and eye drops are all consistent, the negative sample of mixture (containing sugared type and Sugarless type), oral liquid (containing sugared type and Sugarless type), syrup and eye drops by the evidence erigeron breviscapus is all noiseless to measuring, and the retention time that goes out peak number amount and each reference substance in the sample is all consistent, can think that auxiliary material is noiseless to this method.Each organize test determination as a result RSD all less than 2%, the result of the repeatability of the methodology checking of four kinds of preparations and raw material medicinal extract, precision, application of sample recovery test, tolerance test all meets the requirements, therefore can think: the method specificity is strong, precision is high, good reproducibility, accuracy is high, durability good, be applicable to Content of Chlorogenic Acid, the caffeic acid, 1 of these four kinds of erigeron breviscapus liquid preparations of mixture, oral liquid, syrup and eye drops and raw material medicinal extract, 3-O-dicaffeoylquinic acid, these four component concentrations of lamp-dish flower acetic mensuration simultaneously.
Claims (2)
1. 4 kinds of methods that active constituent content detects simultaneously in the fleabane preparation is characterized in that carrying out according to the following steps:
1) preparation of reference substance solution: get chlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 2-60 μ g/ml, contain caffeic acid 3.1-93 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 1.2-36 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 10-300 μ g/ml, for subsequent use;
2) preparation of need testing solution: get 1 mL or 1g fleabane preparation in the 10mL measuring bottle, add purified water 4ml, methanol constant volume shakes up to scale, filters, and gets filtrate as need testing solution;
3) 4 kinds of active constituent content measurings: draw respectively each 10-20 μ L of reference substance solution and need testing solution, in the injection liquid chromatography, measure, the record chromatogram is pressed external standard method with calculated by peak area, and be get final product;
Wherein: the liquid chromatograph octadecylsilane chemically bonded silica is filling agent; In acetonitrile or the methyl alcohol one or more are as mobile phase A, and the aqueous solution of one or more in the formic acid of 0.05%-1.0% or acetic acid or the phosphoric acid is Mobile phase B, by following gradient elution:
Time 0～10min, ratio 7%～9%A;
Time 10～24min, ratio 8%～12%A;
Time 24～37min, ratio 9%～16%A;
Time 37～57min, ratio 10%～24%A;
Time 57～60min, ratio 24%～7%A;
Flow velocity per minute 0.5-2mL; Detect wavelength 327nm; Column temperature 30-40 ℃, chlorogenic acid, caffeic acid, 1, the theoretical cam curve of 3-O-dicaffeoylquinic acid, lamp-dish flower acetic is not less than 5000, with the degree of separation at other peaks should be greater than 1.5.
2. 4 kinds of methods that active constituent content detects simultaneously in the fleabane preparation according to claim 1, it is characterized in that 1) get chlorogenic acid, caffeic acid, 1 in the step, 3-O-dicaffeoylquinic acid, lamp-dish flower acetic reference substance add methyl alcohol dissolving and make every ml and contain chlorogenic acid 12-25 μ g/ml, contain caffeic acid 15-30 μ g/ml, contain 1,3-O-dicaffeoylquinic acid 6-12 μ g/ml, contain the reference substance solution of lamp-dish flower acetic 50-120 μ g/ml, for subsequent use.
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