CN100383522C - Red-bay berry root herb content detecting method - Google Patents

Abstract

The invention relates to a quality control method for natural vegetable drug, especially a testing method for testing myricetrin content. It uses high efficiency liquid phase chromatography to test the content of myricetrin in waxberry root drug. It includes the following steps: testing chromatogram condition and system adaptability, making reference substance liquid, making testing liquid, testing by measurement method, and gaining the result. The invention supplies a content measuring method for waxberry root drug. It could take quality control conveniently, rapidly and accurately.

Description

The content assaying method of Red-bayberry root herb

Technical field

The present invention relates to a kind of method of quality control of natural plant crude drugs, the content analysis that relates to the effective ingredient of medicinal material is measured.

Background technology

Chinese waxmyrtle root is a kind of Chinese medicine medicinal material commonly used, applies in the multiple Chinese patent drug, as thousand purplish red particles, thousand purplish red capsules etc.But as the raw medicinal material in the pharmaceutical field, its content of effective must reach a certain standard, otherwise, can influence its quality greatly at industrial value and made patent medicine.Up to the present, still do not report in the prior art Content Measurement of Effective Ingredient in Happiness method in the Red-bayberry root herb.

Summary of the invention

The objective of the invention is to overcome the defective of prior art, a kind of content assaying method of Red-bayberry root herb is provided.

The content assaying method of Red-bayberry root herb of the present invention is the content with myricetrin in the high effective liquid chromatography for measuring Red-bayberry root herb.

The content assaying method of Red-bayberry root herb of the present invention is made up of following steps:

Chromatographic condition and system suitability test are filling agent with octyl group silane group silica gel; Acetonitrile-0.1% phosphoric acid solution (12: 88) is a moving phase; The detection wavelength is 260nm; Column temperature: 40 ℃; Theoretical cam curve is calculated by myricetrin should be not less than 3000;

Reference substance solution prepares precision, and to take by weighing the myricetrin reference substance an amount of, adds methyl alcohol and make the solution that every 1ml contains 0.05mg, in contrast product solution;

Red-bayberry root herb powder (crossing sieve No. three) 0.1 gram is got in the need testing solution preparation, the accurate title, decide, and puts in the apparatus,Soxhlet's, and it is an amount of to add methyl alcohol, reflux is colourless to methanol extract liquid, put coldly, get methanol extract liquid and place measuring bottle, divide washing container for several times with small amount of methanol, washing lotion is incorporated in the same measuring bottle, add methyl alcohol to scale, shake up, promptly;

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.

Assay research:

It is the assay index that the assay index choice is selected myricetrin.

Instrument and reagent and test material high performance liquid chromatograph: AgiLent1100 series comprises G1314A UV-detector, G1316A column oven, G1313A automatic sampler, G1311A quaternary gradient pump, G1322A vacuum degassing machine, AgiLent ChemStation workstation.

Chromatographic column ZORBAX EcLipse XDB-C8 (4.6 * 150mm, 5-Micron), high purity water adopts distilled water to cross the preparation of MILLIPORE SimpLicity 185 water purification machines; Acetonitrile (HPLC), methyl alcohol (AR), ethanol (AR), phosphoric acid (AR).

Myricetrin reference substance (purity＞98%), Chinese Academy of Sciences Kunming plant provides.

Test condition selects (1) extracting method according to documents and materials and medicinal material characteristics, and employing methyl alcohol is solvent, carries out Soxhlet and extracts.Through the methodology checking, this method is feasible.

(2) assay condition

The selection that detects wavelength is solvent blank with moving phase, in the 200-400nm wavelength coverage, reference substance is scanned, myricetrin has absorption maximum at 260nm, 360nm place, is noiseless peak on the 260nm chromatogram through test detecting wavelength, and the sensitivity that the 260nm place measures is higher, is 260nm so set inspection myricetrin mensuration wavelength.

Being chosen under the identical chromatographic conditions of chromatographic column selects for use following chromatographic column to analyze.The different model chromatographic column to the sample separation degree, that retention time influences difference is big.Factors such as comprehensive degree of separation, peak shape, retention time, octyl silane group silica gel chromatographic column is good than the octadecylsilane chemically bonded silica chromatographic column to the analysis of this sample, so text adopts octyl silane group silica gel chromatographic column as analytical column.

Zorbax

XDB-C18，4.6×250mm，5-Micron

Zorbax XDB-C8，4.6×150mm，5-Micron

Zorbax

XDB-C8，4.6×250mm，5-Micron

100?RP-18e，4.6×250mm，5-Micron

The selection of proportion of mobile phase is according to pertinent literature and data, and the selection acetonitrile-water is a moving phase, adds phosphoric acid and transfers pH=2～3 to reduce the hangover of chromatographic peak in right amount.Different proportions such as (10: 90), (12: 88), (15: 85) have been compared in the mensuration of myricetrin.Factors such as comprehensive degree of separation, peak shape symmetry, retention time show that to measure myricetrin moving phase ratio better for (12: 88) effect, and the symmetry that myricetrin is adjacent the degree of separation of component and peak is all good.

Column temperature carries out stratographic analysis with 25 ℃, 30 ℃, 40 ℃ column temperature respectively under identical chromatographic conditions, with the decline of column temperature, and post voltage rise height, the myricetrin appearance time prolongs, but little to the influence of sample separation degree.Stablizing for the retention time that makes one-tenth swarming to be measured, is 40 ℃ so column temperature is set.

(3) the HPLC collection of illustrative plates of system suitability experimental evidence sample is measured the retention time, half-peak breadth at myricetrin peak etc., and the theoretical plate number average that calculates chromatographic column is greater than 3000, and myricetrin becomes swarming calculating degree of separation with adjacent, all greater than 2.0.Therefore, body specified is in the myricetrin peak, and number of theoretical plate should be greater than 3000.

(4) methodological study

Linear relationship is investigated precision and is taken by weighing myricetrin reference substance 6.25mg and put in the 25ml measuring bottle, adds the dissolving of moving phase solution and is diluted to scale, shakes up, and precision is measured 5ml and put in the 25ml measuring bottle, adds the moving phase solution dilution to scale, shakes up promptly.Accurate respectively reference substance solution 1,2,5,10,15,20,25, the 30 μ L that draw are by above-mentioned chromatographic condition mensuration peak area.With peak area integrated value (mAU*s) is ordinate, and sample size (μ g) is a horizontal ordinate drawing standard curve, calculates regression equation, myricetrin: Y=2387.6X-5.1516, r=0.9999.The result shows that the myricetrin sample size has the good linear relationship (see figure 1) in 0.1042～2.999 μ g scope.

The myricetrin linear test

Myricetrin reference substance solution 5 μ L are drawn in the precision test respectively, repeat sample introduction 5 times, calculate RSD%.Measurement result is 544.98047,546.61377,550.23309,552.69299,560.23633, and RSD% is 0.8%.

Replica test is got same batch sample, and replication is 5 times according to the method described above, and the result is 1.6119%, 1.6122%, 1.6146%, 1.6114%, and RSD% is 1.6%.

Reference substance solution and need testing solution are got in stability test, investigate myricetrin reference substance solution and need testing solution stability respectively at 0,2,6,10,12 hour, and the relative standard deviation of myricetrin peak area integrated value is all less than 2% (see figure 2).

The myricetrin stability test

The accurate respectively sample powder 0.05g that predicts content that takes by weighing of application of sample recovery test, 6 parts, the accurate title, decide, put in the apparatus,Soxhlet's, respectively accurate myricetrin reference substance solution (being mixed with 0.102mg/ml) 7,7,6,6,5, the 5ml of adding with methyl alcohol, it is an amount of to add methyl alcohol, below press the need testing solution preparation method from, " reflux extremely ... " rises, and operates with method.Calculate recovery rate.

Myricetrin recovery test result

Sample determination is measured myricetrin content in 10 batches of Red-bayberry root herb samples according to the method described above, and measurement result is 1.61%, 1.65%, 2.13%, 1.51%, 2.04%, 2.12%, 2.06%, 1.89%, 1.79%, 1.99%.

According to measurement result, this product is pressed dry product and is calculated, and contains myricetrin (C ₂₁H ₂₀O ₁₂) must not be less than 1.50%.

The invention provides a kind of content assaying method of Red-bayberry root herb.System adopts high performance liquid chromatography to measure the content of myricetrin in the Red-bayberry root herb.In order better to control quality of medicinal material, can adopt myricetrin is reference substance, adopts thin-layered chromatography to differentiate Red-bayberry root herb, measures the content of myricetrin in the Red-bayberry root herb again with high performance liquid chromatography.Thereby it is convenient, fast, exactly the quality of Red-bayberry root herb is controlled.

Description of drawings

Fig. 1 is a myricetrin sample size linear relationship chart.

Fig. 2 is myricetrin stability test figure.

Embodiment

Embodiment:

Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia D).

Chromatographic condition and system suitability test are filling agent with octyl group silane group silica gel; Acetonitrile-0.1% (w/w) phosphoric acid solution (12: 88) is a moving phase; The detection wavelength is 260nm; Column temperature: 40 ℃; Theoretical cam curve is calculated by myricetrin should be not less than 3000.

Reference substance solution prepares precision, and to take by weighing the myricetrin reference substance an amount of, adds methyl alcohol and make the solution that every 1ml contains 0.05mg, in contrast product solution.

This product powder (crossing sieve No. three) 0.1 gram is got in the need testing solution preparation, the accurate title, decide, and puts in the apparatus,Soxhlet's, and it is an amount of to add methyl alcohol, reflux is colourless to methanol extract liquid, put coldly, get methanol extract liquid and place the 50ml measuring bottle, divide washing container for several times with small amount of methanol, washing lotion is incorporated in the same measuring bottle, add methyl alcohol to scale, shake up, promptly.

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.

This product is pressed dry product and is calculated, with myricetrin (C ₂₁H ₂₀O ₁₂) meter, must not be less than 1.5%.

More than each reagent be analyze pure, myricetrin purity 〉=95%.

Claims (1)

1. the content assaying method of a Red-bayberry root herb is characterized in that it being the content of using myricetrin in the high effective liquid chromatography for measuring Red-bayberry root herb, it is characterized in that being made up of following steps:

Chromatographic condition and system suitability test are filling agent with octyl group silane group silica gel; Acetonitrile-0.1% phosphoric acid solution is a moving phase at 12: 88; The detection wavelength is 260nm; Column temperature: 40 ℃; Theoretical cam curve is calculated by myricetrin should be not less than 3000;

Reference substance solution prepares precision, and to take by weighing the myricetrin reference substance an amount of, adds methyl alcohol and make the solution that every 1ml contains 0.05mg, in contrast product solution;

Red-bayberry root herb powder 0.1 gram is got in the need testing solution preparation, the accurate title, decide, and puts in the apparatus,Soxhlet's, and it is an amount of to add methyl alcohol, reflux is colourless to methanol extract liquid, put coldly, get methanol extract liquid and place measuring bottle, divide washing container for several times with small amount of methanol, washing lotion is incorporated in the same measuring bottle, add methyl alcohol to scale, shake up, promptly;

Accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.

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