CN102384907A - Method by utilize vanillin-sulfuric acid colorimetry to measure glabridin content - Google Patents
Method by utilize vanillin-sulfuric acid colorimetry to measure glabridin content Download PDFInfo
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Abstract
The invention relates to a method by utilizing vanillin-sulfuric acid colorimetry to measure glabridin content. Collection process of chromogenic reagent absorbency value comprises the steps that: vanillin methanol solution with concentration of 20mg/mL is prepared, sulfuric acid methanol solution with concentration in percentage by volume of 30 percent is prepared, glabridin standard solution with concentration of 0.5mg/mL is prepared, glabridin standard test solution with range of the concentration of 0.01 to 0.5mg/mL is prepared, the absorbency value is collected, the chromogenic reagent is stable and reliable within 60 minutes under the room temperature, so the collection of the absorbency value information of the chromogenic reagent is guaranteed, and finally a linear regression equation is established. Being proved by a recovery test and a repeatability test: the method is stable to analyze, the repeatability conforms to the requirement, the method is proved to be completely reliable, the linear regression equation can provide a quantitative standard to the measurement of the content of the glabridin to be tested, and a test result and test precision, which are identical to the high-efficient liquid-phase spectrum, can be obtained.
Description
Technical field
The invention belongs to plant content determination techniques field, refer more particularly to a kind of method of vanillin-sulfuric acid colorimetric method for determining glabridin content.
Background technology
Glycyrrhiza glabra (
Glycyrrhiza glabra L.) in the isoflavan class that contains of institute spy abbreviate as glabridin (
Glabridin), the isoflavan class has diversified pharmaceutically active and receives much concern.The molecular formula of glabridin is C
20H
20O
4, molecular weight is 324.37.
There is data to show; The glabridin adjuvant in the cosmetics that can be used to whiten through the activity of restraint of tyrosinase and dopachrome interconvertible enzyme TRP-2, hinders 5; The polymerization of 6-dihydroxy indole DHI stops melanic formation, thereby reaches skin whitening effects.
Also have data to show that glabridin has following characteristic:
(1) have stronger antioxidation activity, antioxidant radical ability and vitamin E are close;
(2) have antibiotic, anti-born of the same parents poison, anti-breast cancer cel l proliferation, and significantly hypotensive, reducing blood lipid and stronger neuroprotective effect, its application prospect is very wide.
What present mode about analysis glabridin content adopted is high performance liquid chromatography, and the employed high performance liquid chromatograph of high performance liquid chromatography costs an arm and a leg, and testing cost is high, and detection time is longer, and is also very high to operating personnel's requirement.
Though colourimetry is to measure content of material quantitative analysis method comparatively commonly used at present, yet under the vanillin-sulfuric acid condition, uses the method for colorimetric method for determining glabridin content not appear in the newspapers so far.
Summary of the invention
For addressing the above problem; The invention provides a kind of method of vanillin-sulfuric acid colorimetric method for determining glabridin content; This method is divided into two big steps; First step is the gatherer process of colour developing liquid absorbance information, and second step is that the equation of linear regression of glabridin standard detection solution-mean light absorbency value is set up process, and the required time ratio high performance liquid chromatography of this method shortens greatly; And colour developing liquid is reliable and stable in room temperature 60min, is fit to very much the centralized detecting of a large amount of glabridin content to be measured.
For realizing the foregoing invention purpose, the present invention adopts following technical scheme:
A kind of method of vanillin-sulfuric acid colorimetric method for determining glabridin content, this method is mainly concerned with: standard items purity HPLC>=98% of glabridin, absolute methanol; Mass percent concentration is 98% sulfuric acid, vanillic aldehyde, thermostat water bath; Cuvette, ultraviolet-visible pectrophotometer; This method has resorcinol structure and C ring C through the B ring
2, C
3It is the glabridin of singly-bound and vanillic aldehyde reagent reacts and be to make the reactant liquor colour developing under 98% the sulfuric acid condition at mass percent concentration; Utilize ultraviolet-visible pectrophotometer to measure the absorbance of this colour developing liquid under its maximum absorption wavelength 534nm again; Thereby accomplish the gatherer process of colour developing liquid absorbance information: under said acid condition; The shade of colour developing liquid and the content of glabridin are remarkable linear positive correlation; Meet Lambert-Beer's law; Thereby lay a good foundation for the equation of linear regression foundation of glabridin standard detection solution-mean light absorbency value, said equation of linear regression is that later glabridin Determination on content to be measured provides quantitative criterion.
The gatherer process of said colour developing liquid absorbance information is following:
1. compound concentration is the vanillic aldehyde methanol solution of 20 mg/mL
Take by weighing the vanillic aldehyde of 2.0g with beaker; In beaker, add a small amount of absolute methanol with the dissolving vanillic aldehyde; Vanillic aldehyde methanol solution after the dissolving is transferred in first volumetric flask of 100 mL, used a small amount of absolute methanol rinse beaker 2-3 time again, and the vanillic aldehyde methanol solution after the rinse is also transferred in first volumetric flask; In first volumetric flask, add till absolute methanol solution to the 100 mL scale, obtaining concentration after shaking up is the vanillic aldehyde methanol solution of 20 mg/mL again.
2. the dose volume percent concentration is 30% sulfuric acid methanol solution
Accurately measuring 30 mL mass percent concentrations with transfer pipet is 98% sulfuric acid and second volumetric flask that places 100 mL, in second volumetric flask, adds the absolute methanol of 70 mL again, obtains concentration of volume percent after shaking up and be 30% sulfuric acid methanol solution.
3. compound concentration is the glabridin standard solution of 0.5 mg/mL
Take by weighing the standard items of 12.5 mg glabridins with beaker; In beaker, add the standard items of a small amount of absolute methanol with the dissolving glabridin; The standard items methanol solution of dissolving back glabridin is placed the 3rd volumetric flask of 25mL; Use a small amount of absolute methanol rinse beaker 2-3 time again; And the standard items methanol solution of glabridin after the rinse also transferred in the 3rd volumetric flask, in the 3rd volumetric flask, add till absolute methanol solution to the 25 mL scale again, obtaining concentration after shaking up is the glabridin standard solution of 0.5 mg/mL.
4. prepare the glabridin standard detection solution of 14 parts of concentration ranges at 0.01～0.5 mg/mL
The glabridin standard solution is 14 parts in drawing 3. respectively; The absorption amount of said 14 part of 0.5 mg/mL glabridin standard solution is followed successively by 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.9 mL, 1.1 mL, 1.3 mL, 1.4 mL, 2.0 mL, 2.6 mL, 3.2 mL, 3.8 mL, 4.4 mL and 5.0 mL; And place 14 5 mL volumetric flasks respectively; In 14 5 mL volumetric flasks, add till absolute methanol to the 5 mL scale successively again; Obtain the glabridin standard detection solution that 14 parts of concentration are followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL after shaking up, or abbreviate the glabridin standard detection solution of 14 parts of concentration ranges as at 0.01～0.5 mg/mL.
5. gather absorbance
In the glabridin standard detection solution of 0.01～0.5 mg/mL, respectively draw 0.5 mL and join respectively in the tool plug test tube of 14 10 mL from above-mentioned 14 parts of concentration ranges that obtain 4.; Then to the tool plug test tube of said 14 10 mL add respectively 2.5 mL above-mentioned 1. in the concentration of preparation be vanillic aldehyde methanol solution and 2.5 mL of 20 mg/mL above-mentioned 2. in the concentration of volume percent of preparation be 30% sulfuric acid methanol solution; Then the tool plug test tube of said 14 10 mL is carried out capping and rocks mixing being placed in 20 ℃ of water-baths and taking out behind insulation 20 min; Each tool plug test tube obtains the clarification colour developing liquid of 5.5 mL, obtains 14 parts of said colour developing liquid altogether; From each tool plug test tube, extract less than the said colour developing liquid of 1 ∕ 3 and place cuvette; Utilize ultraviolet-visible pectrophotometer to gather the absorbance information of said colour developing liquid; Using the absorbance information that obtains 14 different numerical value of 14 parts of said colour developing liquid under the same cuvette situation like this; The absorbance information of these 14 different numerical value is called one group, does three groups of parallel experiments under the same conditions altogether to obtain the absorbance information of three groups of different numerical value; Three absorbances that the said colour developing liquid of same concentrations in three groups is gathered carry out arithmetic mean; Promptly obtain the mean light absorbency value of three groups of said colour developing liquid of same concentrations; Can access the mean light absorbency value of the said chromophoric solution of other concentration in three groups in this way; Obtain 14 mean light absorbency values altogether, said 14 mean light absorbency values are corresponding at the glabridin standard detection solution of 0.01～0.5 mg/mL with above-mentioned 14 parts of concentration ranges that obtain in 4..
It is following that the equation of linear regression of said glabridin standard detection solution-mean light absorbency value is set up process:
6. with above-mentioned 4. described in 14 parts of concentration glabridin standard detection solution of being followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL be the x axle; With above-mentioned 5. described in 14 mean light absorbency values be the equation of linear regression that the y axle is set up glabridin standard detection solution-mean light absorbency value; Represent by y=4.4483x and y=1.4208x+0.4013 through the said equation of linear regression of the calculating of EXECL software; The y=4.4483x equation of linear regression that is glabridin standard detection solution when its concentration is 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL and 0.14 mg/mL wherein; Linear concentration range: (0.01-0.14) mg/mL, coefficient R 2=0.9999; The equation of linear regression that y=1.4208x+0.4013 is a glabridin standard detection solution when its concentration is 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL; Linear concentration range: (0.14-0.5) mg/mL, coefficient R 2=0.9957; Can find out: x=0.14 mg/mL is the boundary flex point of y=4.4483x and y=1.4208x+0.4013, and two linearity curves have Different Slope when said boundary flex point.
Because adopt aforesaid technical scheme, the present invention has following superiority:
1, the relative high performance liquid chromatography of the present invention have equally the result accurately, the precision advantages of higher, replaced valuable high performance liquid chromatograph, make that detecting cost significantly reduces.
2, the present invention is based on the B ring and have resorcinol structure and C ring C
2, C
3It is the glabridin of singly-bound reacted and under acid condition, generated the different red complex of the depth with vanillic aldehyde reagent chromophoric solution; Chromophoric solution has the maximum light absorption rate under the 534nm wavelength; Utilize the ultraviolet-visible pectrophotometer collection to measure the absorbance of chromophoric solution; Meet Lambert-Beer's law, set up the equation of linear regression of glabridin content-absorbance on this basis.
3, method of the present invention is easy and simple to handle, quick; Accomplishing whole detecting operation processes only needed less than one hour; Shorten detection time greatly; And colour developing liquid is reliable and stable in room temperature 60min, thereby very is fit to the centralized detecting to a large amount of glabridin content to be measured, is particularly suitable for the detection of dynamic of each production phase in industry extraction and the refining glabridin process.
Description of drawings
Fig. 1It is glabridin standard detection solution concentration-mean light absorbency value typical curve
Embodiment
Vanillic aldehyde claim again vanillic aldehyde (
Vanillin).Document announcement is arranged, and vanillic aldehyde is Chinese crude drug qualitative identification and quantitative test developer commonly used, and this mechanism is based on: C encircles C
2, C
3Between be singly-bound and have resorcinol or the flavone compound of phloroglucin A-ring structure, can with the vanillic aldehyde reagent generation red complex that reacts.
The B ring of glabridin has C in resorcinol structure and the C ring
2, C
3Be singly-bound, thereby glabridin and the vanillic aldehyde generation red complex that under acid condition, also can develop the color, this is for the invention provides basic condition.Though basic condition is set up, the sensitivity of chromogenic reaction is relevant with the concentration of acid in the reaction system, and big its sensitivity of the low then moisture of acid concentration is just poor.Through single factor experiment; It is 98% the concentrated sulphuric acid acid condition as reaction system that the present invention has selected mass percent concentration; Improved the sensitivity and the stability of chromogenic reaction greatly, thereby and mass percent concentration is that 36.6% concentrated hydrochloric acid is because contained humidity causes the sensitivity of chromogenic reaction to reduce improper this reaction system that is used as greatly more greatly.
The standard items of the used glabridin of the present invention are buied from market, standard items purity HPLC >=98% of used glabridin.
The present invention is a kind of method of vanillin-sulfuric acid colorimetric method for determining glabridin content, and this method has resorcinol structure and C ring C through the B ring
2, C
3It is the glabridin of singly-bound and vanillic aldehyde reagent reacts and be to make the reactant liquor colour developing under 98% the sulfuric acid condition at mass percent concentration; Utilize ultraviolet-visible pectrophotometer to measure the absorbance of this colour developing liquid under its maximum absorption wavelength 534nm again; Thereby accomplish the gatherer process of colour developing liquid absorbance information: under said acid condition; The shade of colour developing liquid and the content of glabridin are remarkable linear positive correlation; Meet Lambert-Beer's law; Thereby lay a good foundation for the equation of linear regression foundation of glabridin standard detection solution-mean light absorbency value, said equation of linear regression is that later glabridin Determination on content to be measured provides quantitative criterion.
The gatherer process of colour developing liquid absorbance information according to the invention is following:
1. compound concentration is the vanillic aldehyde methanol solution of 20 mg/mL
Take by weighing the vanillic aldehyde of 2.0g with beaker; In beaker, add a small amount of absolute methanol with the dissolving vanillic aldehyde; Vanillic aldehyde methanol solution after the dissolving is transferred in first volumetric flask of 100 mL, used a small amount of absolute methanol rinse beaker 2-3 time again, and the vanillic aldehyde methanol solution after the rinse is also transferred in first volumetric flask; In first volumetric flask, add till absolute methanol solution to the 100 mL scale, obtaining concentration after shaking up is the vanillic aldehyde methanol solution of 20 mg/mL again.
Attention: the addition of said a small amount of absolute methanol solution is not necessarily equal several times 1., but the total addition of absolute methanol in 1. must be strict controlled in 100 mL.
2. the dose volume percent concentration is 30% sulfuric acid methanol solution
Accurately measuring 30 mL mass percent concentrations with transfer pipet is 98% sulfuric acid and second volumetric flask that places 100 mL, in second volumetric flask, adds the absolute methanol of 70 mL again, obtains concentration of volume percent after shaking up and be 30% sulfuric acid methanol solution.
Concentration of volume percent is that 30% sulfuric acid methanol solution is that chromogenic reaction provides sour environment.
3. compound concentration is the glabridin standard solution of 0.5 mg/mL
Take by weighing the standard items of 12.5 mg glabridins with beaker; In beaker, add the standard items of a small amount of absolute methanol with the dissolving glabridin; The standard items methanol solution of dissolving back glabridin is placed the 3rd volumetric flask of 25mL; Use a small amount of absolute methanol rinse beaker 2-3 time again; And the standard items methanol solution of glabridin after the rinse also transferred in the 3rd volumetric flask, in the 3rd volumetric flask, add till absolute methanol solution to the 25 mL scale again, obtaining concentration after shaking up is the glabridin standard solution of 0.5 mg/mL.
Attention: the addition of said a small amount of absolute methanol is not necessarily equal several times 3., but the total addition of absolute methanol in 3. must be strict controlled in 25 mL.
4. prepare the glabridin standard detection solution of 14 parts of concentration ranges at 0.01～0.5 mg/mL
The glabridin standard solution is 14 parts in drawing 3. respectively; The absorption amount of said 14 part of 0.5 mg/mL glabridin standard solution is followed successively by 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.9 mL, 1.1 mL, 1.3 mL, 1.4 mL, 2.0 mL, 2.6 mL, 3.2 mL, 3.8 mL, 4.4 mL and 5.0 mL; And place 14 5 mL volumetric flasks respectively; In 14 5 mL volumetric flasks, add till absolute methanol to the 5 mL scale successively again; Obtain the glabridin standard detection solution that 14 parts of concentration are followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL after shaking up, or abbreviate the glabridin standard detection solution of 14 parts of concentration ranges as at 0.01～0.5 mg/mL.
Above-mentioned 4. in 14 parts of concentration ranges of reply make not isolabeling at the glabridin standard detection solution of 0.01～0.5 mg/mL, or to 14 5 mL volumetric flasks make not isolabeling also can, in case chaotic.
5. gather absorbance
In the glabridin standard detection solution of 0.01～0.5 mg/mL, respectively draw 0.5 mL and join respectively in the tool plug test tube of 14 10 mL from above-mentioned 14 parts of concentration ranges that obtain 4.; Then to the tool plug test tube of said 14 10 mL add respectively 2.5 mL above-mentioned 1. in the concentration of preparation be vanillic aldehyde methanol solution and 2.5 mL of 20 mg/mL above-mentioned 2. in the concentration of volume percent of preparation be 30% sulfuric acid methanol solution; Then the tool plug test tube of said 14 10 mL is carried out capping and rocks mixing being placed in 20 ℃ of water-baths and taking out behind insulation 20 min; Each tool plug test tube obtains the clarification colour developing liquid of 5.5 mL, obtains 14 parts of said colour developing liquid altogether; From each tool plug test tube, extract less than the said colour developing liquid of 1 ∕ 3 and place cuvette; Utilize ultraviolet-visible pectrophotometer to gather the absorbance information of said colour developing liquid; Using the absorbance information that obtains 14 different numerical value of 14 parts of said colour developing liquid under the same cuvette situation like this; The absorbance information of these 14 different numerical value is called one group, does three groups of parallel experiments under the same conditions altogether to obtain the absorbance information of three groups of different numerical value; Three absorbances that the said colour developing liquid of same concentrations in three groups is gathered carry out arithmetic mean; Promptly obtain the mean light absorbency value of three groups of said colour developing liquid of same concentrations; Can access the mean light absorbency value of the said chromophoric solution of other concentration in three groups in this way; Obtain 14 mean light absorbency values altogether, said 14 mean light absorbency values are corresponding at the glabridin standard detection solution of 0.01～0.5 mg/mL with above-mentioned 14 parts of concentration ranges that obtain in 4..
Above-mentioned cuvette must use same, wants this cuvette of Rapid Cleaning after absorbance information of every collection, and the absorbance of only under same cuvette and same ultraviolet-visible pectrophotometer, being gathered just possibly reduce measuring error to greatest extent.
The model that above-mentioned ultraviolet-visible pectrophotometer uses is the UV-2800 type, and it can accurately measure the absorbance of said chromophoric solution.
Said colour developing liquid is reliable and stable in room temperature 60min, and this provides assurance for the absorbance information of gathering said colour developing liquid.
It is following that the equation of linear regression of glabridin standard detection solution according to the invention-mean light absorbency value is set up process:
6. with above-mentioned 4. described in 14 parts of concentration glabridin standard detection solution of being followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL be the x axle; With above-mentioned 5. described in 14 mean light absorbency values be the equation of linear regression that the y axle is set up glabridin standard detection solution-mean light absorbency value; Represent by y=4.4483x and y=1.4208x+0.4013 through the said equation of linear regression of the calculating of EXECL software; The y=4.4483x equation of linear regression that is glabridin standard detection solution when its concentration is 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL and 0.14 mg/mL wherein; Linear concentration range: (0.01-0.14) mg/mL, coefficient R 2=0.9999; The y=1.4208x+0.4013 equation of linear regression that is glabridin standard detection solution when its concentration is 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL wherein; Linear concentration range: (0.14-0.5) mg/mL, coefficient R 2=0.9957; Can find out: x=0.14 mg/mL is the boundary flex point of y=4.4483x and y=1.4208x+0.4013, and two linearity curves have Different Slope when said boundary flex point.
Y=4.4483x and y=1.4208x+0.4013 are that later glabridin Determination on content to be measured provides quantitative criterion.
Fig. 1 is glabridin standard detection solution-mean light absorbency value typical curve of setting up according to said equation of linear regression; Horizontal ordinate is that the x axle is the concentration range of glabridin standard detection solution among the figure, and concentration among the figure (mg/mL) is represented the concentration of glabridin standard detection solution; Ordinate is that the y axle is the scope of mean light absorbency value among the figure, and A represents the mean light absorbency value among the figure.
The application example of said equation of linear regression
From the glabridin subtractive process, randomly draw its sample A, sample A is become sample A solution to be measured by above-mentioned 3. arrangements of steps, and then utilize the concentration of said equation of linear regression calculation sample A solution to be measured.
The sample A solution to be measured of 0.5 mL is joined in the tool plug test tube of 10 mL; In tool plug test tube, adding 2.5 mL concentration successively is that 20 mg/mL vanillic aldehyde methanol solutions and 2.5 mL concentration of volume percent are 30% sulfuric acid methanol solution; Tool plug test tube is carried out capping and rocks mixing; Take out after then tool plug test tube being placed 20 ℃ of water-bath insulation 20 min, obtain the clarification colour developing liquid of sample A solution to be measured.
From above-mentioned tool plug test tube, extract less than the said colour developing liquid of 1 ∕ 3 and place cuvette; Utilize ultraviolet-visible pectrophotometer to gather the absorbance information of said colour developing liquid, do three groups of parallel experiments under the same conditions altogether to obtain the absorbance information of three groups of different numerical value.The absorbance of three groups of different numerical value is carried out arithmetic mean; Obtain the mean light absorbency value of the said colour developing liquid of sample A solution to be measured; With mean light absorbency value substitution equation of linear regression, the concentration that calculates glabridin in the sample A solution to be measured is 0.124 mg/mL.
The concentration that can calculate glabridin in the sample B solution to be measured according to aforesaid way equally is 0.221 mg/mL.
Recovery experiment
1. recovery experiment 1
Respectively get 5mL concentration and be six parts of the sample A solution to be measured of 0.124 mg/mL and place six 10 mL test tubes respectively; In six 10 mL test tubes, add glabridin standard solution 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL that concentration is 0.5 mg/mL more successively; Six 10 mL test tubes are made six addition marks; In six test tubes of making mark, add absolute methanol to 10mL scale respectively then and end, fully obtain the sample A solution of six parts of dilutions behind the mixing.
The former content value of glabridin is 0.124mg/mL * 5mL=0.62 mg in the above-mentioned six duplicate samples A solution to be measured, and the adding value of glabridin is respectively 0 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg and 0.5 mg in the glabridin standard solution.
Get each 0.5mL of sample A solution of said six parts of dilutions; By above-mentioned 5. step and can calculate the glabridin concentration in the sample A solution of said six parts of dilutions respectively according to said equation of linear regression; And calculating total measured value of the glabridin in the sample A solution of said six parts of dilutions, total measured value is respectively 0.620 mg, 0.721 mg, 0.819 mg, 0.92 mg, 1.022 mg and 1.121 mg.
Deduct the measured value that said former content value is glabridin addition in the glabridin standard solution with said total measured value; The ratio of said measured value and adding value is the recovery; Obtain the standard deviation between the said recovery value with EXECL software, the ratio of said standard deviation and recovery arithmetic mean is exactly the relative standard deviation of recovery test.
Concrete outcome sees following table for details:
2. recovery test 2
Can make the recovery test of sample B solution to be measured as stated above.Concrete outcome sees the following form:
The reappearance test
Reappearance test 1:
Respectively get 0.5mL concentration and be eight parts of the sample A solution to be measured of 0.124 mg/mL and place eight 10 mL tool plug test tubes respectively; By above-mentioned 5. step and can calculate the measured value of glabridin concentration in the said eight duplicate samples A solution to be measured respectively according to said equation of linear regression; Try to achieve eight standard deviations between the measured value with EXECL software; The ratio of said standard deviation and measured value arithmetic mean is exactly the relative standard deviation of reappearance test, and said relative standard deviation is 1.72%.
Reappearance test 2:
Record the measured value of glabridin concentration in the eight duplicate samples B solution to be measured as stated above, and finally to try to achieve relative standard deviation be 1.24%.
The concrete outcome of above-mentioned reappearance test 1 and 2 is summed up and is seen the following form:
Show that through concrete example application and recovery test and reappearance test the testing sample of randomly drawing according to the practical implementation step, all can accurately determine the content of glabridin in the testing sample through the inventive method.Recovery experiment and reappearance result of experiment have explained that methods analyst of the present invention is stable, and reappearance meets the requirements, and prove that method of the present invention is that said equation of linear regression is that later glabridin Determination on content to be measured provides quantitative criterion fully reliably.
In addition in order to verify reliability of the present invention; Through high performance liquid chromatography method of the present invention is verified; Net result shows: method of the present invention has accuracy, and method of the present invention can obtain testing result identical with high performance liquid chromatography and precision.
Need to prove at last:
1, entire reaction course of the present invention should be in anhydrous state;
2, operating process of the present invention is fast and convenient, the result is accurate, and the said chromophoric solution that obtains is stablized desirable in 60min;
3, equation of linear regression is at the present invention's down gained that imposes a condition, and is equal to replacement according to method of the present invention and all should be encompassed among the technical scheme of the present invention.
Claims (1)
1. the method for a vanillin-sulfuric acid colorimetric method for determining glabridin content, this method is mainly concerned with: standard items purity HPLC>=98% of glabridin, absolute methanol; Mass percent concentration is 98% sulfuric acid, vanillic aldehyde, thermostat water bath; Cuvette, ultraviolet-visible pectrophotometer; This method has resorcinol structure and C ring C through the B ring
2, C
3It is the glabridin of singly-bound and vanillic aldehyde reagent reacts and be to make the reactant liquor colour developing under 98% the sulfuric acid condition at mass percent concentration; Utilize ultraviolet-visible pectrophotometer to measure the absorbance of this colour developing liquid under its maximum absorption wavelength 534nm again; Thereby accomplish the gatherer process of colour developing liquid absorbance information: under said acid condition; The shade of colour developing liquid and the content of glabridin are remarkable linear positive correlation; Meet Lambert-Beer's law; Thereby lay a good foundation for the equation of linear regression foundation of glabridin standard detection solution-mean light absorbency value, said equation of linear regression is that later glabridin Determination on content to be measured provides quantitative criterion; It is characterized in that:
The gatherer process of said colour developing liquid absorbance information is following:
1. compound concentration is the vanillic aldehyde methanol solution of 20 mg/mL
Take by weighing the vanillic aldehyde of 2.0g with beaker; In beaker, add a small amount of absolute methanol with the dissolving vanillic aldehyde; Vanillic aldehyde methanol solution after the dissolving is transferred in first volumetric flask of 100 mL, used a small amount of absolute methanol rinse beaker 2-3 time again, and the vanillic aldehyde methanol solution after the rinse is also transferred in first volumetric flask; In first volumetric flask, add till absolute methanol solution to the 100 mL scale, obtaining concentration after shaking up is the vanillic aldehyde methanol solution of 20 mg/mL again;
2. the dose volume percent concentration is 30% sulfuric acid methanol solution
Accurately measuring 30 mL mass percent concentrations with transfer pipet is 98% sulfuric acid and second volumetric flask that places 100 mL, in second volumetric flask, adds the absolute methanol of 70 mL again, obtains concentration of volume percent after shaking up and be 30% sulfuric acid methanol solution;
3. compound concentration is the glabridin standard solution of 0.5 mg/mL
Take by weighing the standard items of 12.5 mg glabridins with beaker; In beaker, add the standard items of a small amount of absolute methanol with the dissolving glabridin; The standard items methanol solution of dissolving back glabridin is placed the 3rd volumetric flask of 25mL; Use a small amount of absolute methanol rinse beaker 2-3 time again; And the standard items methanol solution of glabridin after the rinse also transferred in the 3rd volumetric flask, in the 3rd volumetric flask, add till absolute methanol solution to the 25 mL scale again, obtaining concentration after shaking up is the glabridin standard solution of 0.5 mg/mL;
4. prepare the glabridin standard detection solution of 14 parts of concentration ranges at 0.01～0.5 mg/mL
The glabridin standard solution is 14 parts in drawing 3. respectively; The absorption amount of said 14 part of 0.5 mg/mL glabridin standard solution is followed successively by 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.9 mL, 1.1 mL, 1.3 mL, 1.4 mL, 2.0 mL, 2.6 mL, 3.2 mL, 3.8 mL, 4.4 mL and 5.0 mL; And place 14 5 mL volumetric flasks respectively; In 14 5 mL volumetric flasks, add till absolute methanol to the 5 mL scale successively again; Obtain the glabridin standard detection solution that 14 parts of concentration are followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL after shaking up, or abbreviate the glabridin standard detection solution of 14 parts of concentration ranges as at 0.01～0.5 mg/mL;
5. gather absorbance
In the glabridin standard detection solution of 0.01～0.5 mg/mL, respectively draw 0.5 mL and join respectively in the tool plug test tube of 14 10 mL from above-mentioned 14 parts of concentration ranges that obtain 4.; Then to the tool plug test tube of said 14 10 mL add respectively 2.5 mL above-mentioned 1. in the concentration of preparation be vanillic aldehyde methanol solution and 2.5 mL of 20 mg/mL above-mentioned 2. in the concentration of volume percent of preparation be 30% sulfuric acid methanol solution; Then the tool plug test tube of said 14 10 mL is carried out capping and rocks mixing being placed in 20 ℃ of water-baths and taking out behind insulation 20 min; Each tool plug test tube obtains the clarification colour developing liquid of 5.5 mL, obtains 14 parts of said colour developing liquid altogether; From each tool plug test tube, extract less than the said colour developing liquid of 1 ∕ 3 and place cuvette; Utilize ultraviolet-visible pectrophotometer to gather the absorbance information of said colour developing liquid; Using the absorbance information that obtains 14 different numerical value of 14 parts of said colour developing liquid under the same cuvette situation like this; The absorbance information of these 14 different numerical value is called one group, does three groups of parallel experiments under the same conditions altogether to obtain the absorbance information of three groups of different numerical value; Three absorbances that the said colour developing liquid of same concentrations in three groups is gathered carry out arithmetic mean; Promptly obtain the mean light absorbency value of three groups of said colour developing liquid of same concentrations; Can access the mean light absorbency value of the said chromophoric solution of other concentration in three groups in this way; Obtain 14 mean light absorbency values altogether, said 14 mean light absorbency values are corresponding at the glabridin standard detection solution of 0.01～0.5 mg/mL with above-mentioned 14 parts of concentration ranges that obtain in 4.;
It is following that the equation of linear regression of said glabridin standard detection solution-mean light absorbency value is set up process:
6. with above-mentioned 4. described in 14 parts of concentration glabridin standard detection solution of being followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL be the x axle; With above-mentioned 5. described in 14 mean light absorbency values be the equation of linear regression that the y axle is set up glabridin standard detection solution-mean light absorbency value; Represent by y=4.4483x and y=1.4208x+0.4013 through the said equation of linear regression of the calculating of EXECL software; The y=4.4483x equation of linear regression that is glabridin standard detection solution when its concentration is 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL and 0.14 mg/mL wherein; Linear concentration range: (0.01-0.14) mg/mL, coefficient R 2=0.9999; The equation of linear regression that y=1.4208x+0.4013 is a glabridin standard detection solution when its concentration is 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL; Linear concentration range: (0.14-0.5) mg/mL, coefficient R 2=0.9957; Can find out: x=0.14 mg/mL is the boundary flex point of y=4.4483x and y=1.4208x+0.4013, and two linearity curves have Different Slope when said boundary flex point.
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| CN102621089A (en) * | 2012-04-05 | 2012-08-01 | 天津大学 | Method for determining glycyrrhizic acid content in extract after polysaccharide extraction of glycyrrhiza by virtue of vanillin-sulphuric acid |
| CN108918449A (en) * | 2018-07-13 | 2018-11-30 | 河南工业大学 | A kind of paddy xanthochromia degree detection method based on UV-VIS spectrophotometry |
| CN111579507A (en) * | 2020-05-22 | 2020-08-25 | 国珍健康科技（北京）有限公司 | Sulfuric acid-vanillin method for detecting content of procyanidine |
| CN113834790A (en) * | 2021-09-07 | 2021-12-24 | 湘潭大学 | Method for detecting camellia oil doping |
| CN114527117A (en) * | 2022-02-23 | 2022-05-24 | 云南省烟草质量监督检测站 | Method for measuring contents of glucose and xylose in sugar solution and tobacco leaching solution |
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