CN105929039A - Method using HPLC to measure ursolic acid content of haw lozenge - Google Patents
Method using HPLC to measure ursolic acid content of haw lozenge Download PDFInfo
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- CN105929039A CN105929039A CN201610234951.4A CN201610234951A CN105929039A CN 105929039 A CN105929039 A CN 105929039A CN 201610234951 A CN201610234951 A CN 201610234951A CN 105929039 A CN105929039 A CN 105929039A
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- ursolic acid
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a method using HPLC to measure the ursolic acid content of a haw lozenge. The method has the advantages of simple operation and high specificity. The method comprises the following steps: preparing a control solution, preparing a test solution, precisely measuring 4 [mu]L of control solution and 4 [mu]L of test solution, injecting measured control solution and test solution into a high performance liquid chromatographic instrument, measuring the integral value of peak area, calculating the content by an external standard method according to the peak area, and drawing a standard curve. The invention belongs to the technical field of chemical detection.
Description
Technical field
The invention discloses the assay method of a kind of ursolic acid, specifically, be that a kind of HPLC measures Fructus Crataegi
The method of buccal tablet ursolic acid, belongs to chemical extraction technical field.
Background technology
Hawthorn buccal tablet is made up of Fructus Crataegi, white sugar.Rich in compositions such as ursolic acids in Fructus Crataegi, there is depressed liver-energy dispersing and QI regulating,
Blood circulation promoting and blood stasis dispelling, the effect such as aid digestion, it is mainly used in treating the symptom such as fatty liver and hepatic fibrosis.This research is adopted
By HPLC method, index components ursolic acid in three arrow grass and hall hawthorn buccal tablet is carried out assay.Through consulting literary composition
Offer, not yet have the report that its composition ursolic acid is done assay.
Summary of the invention
For the problems referred to above, it is an object of the invention to provide a kind of easy and simple to handle, the HPLC that specificity is strong surveys
The method determining hawthorn buccal tablet ursolic acid.
For solving the problems referred to above, the technical scheme that the present invention provides is such that
A kind of HPLC measures the method for hawthorn buccal tablet ursolic acid, comprises the steps: successively
1) preparation of reference substance solution
Precision weighs ursolic acid reference substance 1.078mg and puts in 10ml measuring bottle, adds methanol and dissolves and be diluted to scale,
Make every 1ml solution containing 107.8 μ g；
2) preparation of need testing solution
Precision measures this product powder 3g, is placed in 50ml conical flask, the accurate 20ml methanol that adds, weighed heavy
Amount, ultrasonic 20min, supplies weight with methanol after placing room temperature, shakes up, filter with microporous filter membrane, take continuous filter
Liquid, to obtain final product；
3) precision draws reference substance and each 4 μ l of need testing solution respectively, injects high performance liquid chromatograph, measures
Integrating peak areas value, by external standard method with calculated by peak area, to obtain final product.
4) preparation of standard curve
Precision weighs ursolic acid reference substance 10.78mg, puts in 10ml measuring bottle, adds methanol and makes dissolving and be diluted to
Scale is as reference substance storing solution, and accurate absorption reference substance storing solution, 1ml puts in 10ml volumetric flask, adds first
Alcohol makes dissolving and is diluted to scale, shakes up, and filters with microporous filter membrane (0.22 μm), takes subsequent filtrate, by dividing
Other sample introduction draws above-mentioned reference substance solution 1,5,8,10,20 μ l respectively, injects high performance liquid chromatograph,
Measuring, with integrating peak areas value as vertical coordinate, ursolic acid reference substance sample size is that abscissa draws standard curve,
Linear equation: Y=1941.82X+0.185, r=0.9999.
Further, above-mentioned a kind of HPLC measures the method for hawthorn buccal tablet ursolic acid, described flowing phase
For acetonitrile-0.1% phosphoric acid 68:32 by volume.
Further, above-mentioned a kind of HPLC measures the method for hawthorn buccal tablet ursolic acid, described detection ripple
Long 210nm.
Compared with prior art, the technical scheme detection method that the present invention provides is simple, stable, repeatable
Good, specificity is strong, is conducive to controlling the quality of short, bristly hair or beard buccal tablet.
Accompanying drawing explanation
Fig. 1 is ursolic acid reference substance HPLC chromatogram；
Fig. 2 is sample HPLC chromatogram；
Fig. 3 is negative sample HPLC chromatogram.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the claim of the present invention is described in further detail, but not
Constitute any limitation of the invention, anyone limited number of time done in the claims in the present invention protection domain
Amendment, still within the claims of the present invention.
The percentage concentration that the present invention relates to, is not particularly illustrated, and liquid is volumetric concentration, solute
Finger mass concentration for solid.
Embodiment 1
1 instrument and reagent
1.1 instrument Aglient 1260 biologically inert quaternary high performance liquid chromatographs, Germany prunus mume (sieb.) sieb.et zucc. Teller-Tuo Li
Many XA205DU electronic analytical balance.
1.2 samples: grass and hall hawthorn buccal tablet: health product factory of Sanjian Pharm. Co., Ltd., Wuzhou, Guangxi produces and (criticizes
Numbers 20120816,20121217,20120320)；Negative sample: lack Fructus Crataegi by prescription and prepare by technique.Bears
Fruit acid reference substance is purchased from Nat'l Pharmaceutical & Biological Products Control Institute, and (lot number: 110742-200517, for assay
With)；Other chemical reagent are analytical pure.
2 methods and result
2.1 chromatographic condition chromatographic column phenomenex Kinete x2.6u XB-C18100A post (100mm
× 4.6mm, 5 μm)；Acetonitrile-0.1% phosphate aqueous solution (68:32) is flowing phase；Flow velocity is 1.0ml.min-1；
Detection wavelength is 210nm.
The preparation precision of 2.2 reference substance solution weighs ursolic acid reference substance 1.078mg and puts 10ml measuring bottle
In, add methanol and dissolve and be diluted to scale, make every 1ml solution containing 107.8 μ g.
The preparation precision of 2.3 need testing solutions measures this product powder 3g, is placed in 50ml conical flask, essence
Close addition 20ml methanol, weighed weight, ultrasonic 20min, supply weight with methanol after placing room temperature, shake up,
Filter with microporous filter membrane (0.22 μm), take subsequent filtrate, to obtain final product.
2.4 algoscopys precision respectively draws reference substance and each 4 μ l of need testing solution, injects high-efficient liquid phase color
Spectrometer, measures integrating peak areas value.By external standard method with calculated by peak area, to obtain final product.
2.5 specificities are investigated and are taken negative sample, by the 2.3 negative need testing solutions of preparation, by 2.1 chromatographs
Condition measures.Result shows that negative control is noiseless under this chromatographic condition, and result is shown in Fig. 1 to Fig. 3.
The preparation precision of 2.6 standard curves weighs ursolic acid reference substance 10.78mg, puts in 10ml measuring bottle,
Add methanol make dissolving and be diluted to scale (concentration is: 1.078mg.mL-1) as reference substance storing solution, essence
Close absorption reference substance storing solution, 1ml puts in 10ml volumetric flask, adds methanol and makes dissolving and to be diluted to scale (dense
Degree is: 0.1078mg.mL-1), shake up, filter with microporous filter membrane (0.22 μm), take subsequent filtrate, press
2.1 chromatographic conditions sample introduction respectively draws above-mentioned reference substance solution 1,5,8,10,20 μ l respectively, injects efficiently
Chromatograph of liquid, measures, with integrating peak areas value as vertical coordinate (Y), and ursolic acid reference substance sample size (mg)
Draw standard curve for abscissa (X), obtain linear equation: Y=1941.82X+0.185 (r=0.9999)
Result shows: ursolic acid is in the range of 13.5～53.9ng, and sample size and integrating peak areas value are good line
Sexual relationship.
2.7 Precision Experiments take with a need testing solution, by 2.1 chromatographic conditions, and continuous sample introduction 5 times,
Measure integrating peak areas value, RSD=0.3%.Show that instrument precision is good.
2.8 stability experiments take with a sample solution, place 0,2,4,6,8,10h, by 2.1
Chromatographic condition, measures integrating peak areas value, respectively by external standard method with calculated by peak area RSD for 0.8%.Result
Show that determinand is stable in 10 hours.
2.9 repeated experiment precisions weigh with batch of sample (lot number: 20120816), molten by 2.3 test samples
The preparation method test of liquid, operation repetitive 5 parts, measure by the 2.1 nearly samples of chromatographic condition, by external standard method with face, peak
Long-pending calculating, result average content is 5.3mg.g-1, RSD=0.6%.Result shows that this method repeatability is good.
2.10 average recovery experiment precisions weigh the hawthorn buccal tablet (lot number: 20120816) measuring content,
(ursolic acid content is 5.3mg.g-1) 1.0g, parallel sampling 9 parts.The most above-mentioned concentration is 1.078mg.mL-1
Ursolic acid reference substance solution 0.5,0.8,1ml, each sample-adding amount is parallel does 3 parts.Molten by 2.3 test samples
The preparation method of liquid is prepared need testing solution, is measured by 2.1 chromatographic conditions, by external standard method with calculated by peak area
The response rate, average recovery rate is 97.6% (n=9), RSD=0.9%.
2.11 sample sizes measure prepares need testing solution by the preparation method of 2.3 need testing solutions, and 2.1
Chromatographic condition, 2.4 algoscopys determine the ursolic acid content in this product 2 batch sample, the results are shown in Table 2.
Table 2, sample determination result
Claims (3)
1. the method that a HPLC measures hawthorn buccal tablet ursolic acid, it is characterised in that include following step successively
Rapid:
1) preparation of reference substance solution
Precision weighs ursolic acid reference substance 1.078mg and puts in 10ml measuring bottle, adds methanol and dissolves and be diluted to scale,
Make every 1ml solution containing 107.8 μ g；
2) preparation of need testing solution
Precision measures this product powder 3g, is placed in 50ml conical flask, the accurate 20ml methanol that adds, weighed heavy
Amount, ultrasonic 20min, supplies weight with methanol after placing room temperature, shakes up, filter with microporous filter membrane, take continuous filter
Liquid, to obtain final product；
3) precision draws reference substance and each 4 μ l of need testing solution respectively, injects high performance liquid chromatograph, measures
Integrating peak areas value, by external standard method with calculated by peak area, to obtain final product.
4) preparation of standard curve
Precision weighs ursolic acid reference substance 10.78mg, puts in 10ml measuring bottle, adds methanol and makes dissolving and be diluted to
Scale is as reference substance storing solution, and accurate absorption reference substance storing solution, 1ml puts in 10ml volumetric flask, adds first
Alcohol makes dissolving and is diluted to scale, shakes up, and filters with microporous filter membrane (0.22 μm), takes subsequent filtrate, by dividing
Other sample introduction draws above-mentioned reference substance solution 1,5,8,10,20 μ l respectively, injects high performance liquid chromatograph,
Measuring, with integrating peak areas value as vertical coordinate, ursolic acid reference substance sample size is that abscissa draws standard curve,
Linear equation: Y=1941.82X+0.185, r=0.9999.
A kind of HPLC the most according to claim 1 measures the method for hawthorn buccal tablet ursolic acid, its feature
Being, described flowing is acetonitrile-0.1% phosphoric acid 68:32 by volume mutually.
A kind of HPLC the most according to claim 1 measures the method for hawthorn buccal tablet ursolic acid, its feature
It is, described detection wavelength 210nm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610234951.4A CN105929039A (en) | 2016-04-15 | 2016-04-15 | Method using HPLC to measure ursolic acid content of haw lozenge |
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| CN201610234951.4A CN105929039A (en) | 2016-04-15 | 2016-04-15 | Method using HPLC to measure ursolic acid content of haw lozenge |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109856306A (en) * | 2019-02-28 | 2019-06-07 | 广东省第二中医院（广东省中医药工程技术研究院） | A kind of method that UHPLC-MS-MS measures ursolic acid in water banyan |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104007223A (en) * | 2014-06-10 | 2014-08-27 | 山西振东开元制药有限公司 | Quality control method of hawthorn middle burner adjusting preparation |
| CN104749281A (en) * | 2015-04-03 | 2015-07-01 | 山东宏济堂制药集团有限公司 | Detection method of children digestion drugs |
-
2016
- 2016-04-15 CN CN201610234951.4A patent/CN105929039A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104007223A (en) * | 2014-06-10 | 2014-08-27 | 山西振东开元制药有限公司 | Quality control method of hawthorn middle burner adjusting preparation |
| CN104749281A (en) * | 2015-04-03 | 2015-07-01 | 山东宏济堂制药集团有限公司 | Detection method of children digestion drugs |
Non-Patent Citations (2)
| Title |
|---|
| 丁银花等: "不同产地山楂中熊果酸和齐墩果酸量的差异分析", 《中草药》 * |
| 曾彬等: "高效液相色谱法测定山楂药材中熊果酸的含量", 《亚太传统医药》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109856306A (en) * | 2019-02-28 | 2019-06-07 | 广东省第二中医院（广东省中医药工程技术研究院） | A kind of method that UHPLC-MS-MS measures ursolic acid in water banyan |
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Application publication date: 20160907 |