CN107807182B - Method for measuring content of ganoderic acid A in ganoderma lucidum syrup - Google Patents
Method for measuring content of ganoderic acid A in ganoderma lucidum syrup Download PDFInfo
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Abstract
The invention relates to a method for measuring the content of ganoderic acid A in ganoderma syrup, which comprises the steps of preparing a test solution, preparing a reference solution and measuring by high performance liquid chromatography. By adopting the content determination method provided by the invention, during determination, the detection baseline is stable, the separation degree is high, the detection accuracy is high, and the durability is good; the method shortens the content determination time, stably improves the detection efficiency, reduces the dosage of toxic chemical reagents, and is more economic and environment-friendly; the invention firstly establishes a high performance liquid chromatography method for determining the content of ganoderic acid A in the ganoderma lucidum syrup, and provides an effective quality detection method for better improving the product quality of the ganoderma lucidum syrup.
Description
Technical Field
The invention relates to a content determination method of Chinese patent medicines, in particular to a content determination method of an active ingredient ganoderic acid A in ganoderma lucidum syrup.
Background
The Ganoderma syrup is prepared from Ganoderma, has effects in nourishing heart, tranquilizing mind, invigorating spleen and regulating stomach function, and can be used for adjuvant treatment of palpitation, insomnia, anorexia, neurasthenia, hyperlipidemia, coronary heart disease, and chronic bronchitis. Ganoderic acid is one of main active ingredients in Ganoderma, and has effects of regulating human glucose metabolism, protecting liver, resisting oxidation, and improving immunity.
The original standard of ganoderma lucidum syrup is collected on page 122 of the seventeenth book Chengfang preparation, and the standard does not disclose the content determination method of the active ingredients, but can not scientifically control the quality of ganoderma lucidum syrup products.
A method for measuring the content of ganoderma lucidum syrup is disclosed in Jilin province drug standard, and comprises the following specific steps: 3g of the product is taken and precisely weighed, the product is placed in a 200mL Kjeldahl flask, 10 parts of potassium sulfate and 1 part of copper sulfate mixed powder are added into the product 1.5g, 10mL of concentrated sulfuric acid are added, 1mL of hydrogen peroxide is carefully added along the wall, and the mixture is heated by direct fire until complete digestion (simultaneously blank) and then is completely transferred into a half-trace nitrogen determination device. And putting another 20mL of boric acid absorption liquid into a 250mL conical flask, inserting the lower opening of the condenser into the boric acid absorption liquid, starting to heat the distillation flask, adding 30mL of 50% sodium hydroxide solution into the Kjeldahl flask through the upper funnel, stopping distillation when the outflow liquid reaches about 80-100 mL, washing the tail end of the condensation tube with a small amount of distilled water, taking down the absorption liquid, and titrating the absorption liquid to grey purple with N/10 sulfuric acid solution to obtain the product (each mLN/10 sulfuric acid solution is equivalent to 1.401mg of N). The total nitrogen content should be above 0.04%.
The above standards disclose a method for measuring the content of N in the ganoderma lucidum syrup, which cannot use the major functional component, i.e., ganoderic acid A with higher content in ganoderma lucidum triterpene, as a measurement index, so that the quality of the ganoderma lucidum syrup product cannot be better controlled.
The agricultural industry standard NY/T2278-2012 of the people's republic of China discloses a method for determining the content of ganoderic acid in a ganoderma lucidum product by high performance liquid chromatography, which comprises the following specific steps: taking not less than 200g of representative ganoderma lucidum fruiting body, crushing, filling into a sealed container, and storing at 0-20 ℃ for later use. Weighing 0.5g of Ganoderma lucidum powder in a 50mL centrifuge tube, adding 25mL of methanol, shaking, ultrasonically extracting for 30min, filtering the extract by filter paper, placing 5mL of the filtrate in a 10mL graduated test tube, drying at about 50 ℃ with nitrogen, dissolving the residue with 1mL of methanol, filtering, and analyzing by high performance liquid chromatography.
High performance liquid chromatography conditions: a C18 column, 250mm × 4.6mm (i.d.),5 μm, or equivalent size chromatography column; mobile phase: acetonitrile-0.1% aqueous formic acid 30: 70; flow rate 1mL/min, column temperature: at 40 deg.C, wavelength of 258nm, and sample size of 20 μ L.
The method has poor separation degree of ganoderic acid A when actually detecting the content of ganoderic acid A in the ganoderma lucidum syrup. And the temperature of the column is higher, so that the damage to the chromatographic column is larger.
In order to better detect the content of the active ingredient ganoderic acid A in the ganoderma lucidum syrup and improve the quality of the ganoderma lucidum syrup, researches are imperatively carried out to provide a content detection method of the ganoderma lucidum syrup, which is simple and convenient to operate, strong in specificity, high in separation degree, good in durability, efficient and environment-friendly.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a content determination method for an active ingredient ganoderic acid A in ganoderma lucidum syrup, and the content determination method provided by the invention has the advantages of stable detection baseline, high separation degree, high detection accuracy and good durability during detection; the retention time of the active ingredient ganoderic acid A is short, the content determination time can be shortened, the detection efficiency is stably improved, the dosage of toxic chemical reagents is reduced, and the method is more economic and environment-friendly; the invention firstly establishes a high performance liquid chromatography method for detecting the content of ganoderic acid A in the ganoderma lucidum syrup, and provides an effective quality detection method for better improving the product quality of the ganoderma lucidum syrup.
Specifically, the invention provides a method for measuring the content of an active ingredient ganoderic acid A in ganoderma lucidum syrup, which comprises the steps of preparation of a test solution, preparation of a reference solution and high performance liquid chromatography measurement.
Preferably, the chromatographic conditions determined by high performance liquid chromatography are as follows: an insertstation C18 chromatography column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05 percent to 0.5 percent, and the gradient elution method is adopted; the detection wavelength is 200-400 nm, the column temperature is 35-40 ℃, and the flow rate is 0.5-2.0 mL/min.
Among the chromatographic conditions, the InertSustain C18 column has a model size of 250X 4.6mm,5 μm, Shimadzu column, in order to achieve more excellent separation effect.
Preferably, in the chromatographic conditions, the mobile phase B is 0.1% by volume of aqueous formic acid solution, and the flow rate is 1.0 mL/min.
As a preferred embodiment, the gradient elution method is: the gradient elution method comprises the following steps: 0-5 min, 2% -10% A; 5-7 min, 2% -10% → 30% -50% A; 7-30 min, 30% -50% A; 30-31 min, 30% -50% → 70% -90% A; 31-45 min, 70% -90% A.
In order to better achieve the separation of the effective components, it is further preferred that the gradient elution method is: 0-5 min, 5% A; 5-7 min, 5% → 36% A; 7-30 min, 36% A; 30-31 min, 36% → 85% A; 31-45 min, 85% A.
Preferably, in the chromatographic condition, the detection wavelength is 258nm, and under the condition of the detection wavelength, the absorbance of the effective component ganoderic acid A is the highest.
Preferably, in the chromatographic condition, the column temperature is 35 ℃, under the column temperature condition, the measured content is higher in accuracy and more stable, and the chromatographic column is not easily damaged under the column temperature condition.
Preferably, the preparation method of the test solution comprises the following steps: precisely measuring 10-30 mL of ganoderma lucidum syrup, placing the ganoderma lucidum syrup into a conical flask with a plug, extracting with 30-60 mL of ethyl acetate, repeatedly extracting for 2-4 times, combining upper layer liquid, evaporating to dryness, dissolving residues with methanol, fixing the volume in a volumetric flask with 5-10 mL, shaking up, and filtering with a microporous filter membrane to obtain a sample solution;
more preferably, the preparation method of the test solution is as follows: precisely measuring 20mL of ganoderma lucidum syrup, placing the ganoderma lucidum syrup into a conical flask with a plug, extracting with 50mL of ethyl acetate, repeatedly extracting for 3 times, taking the upper layer liquid, evaporating to dryness, dissolving the residue with methanol, fixing the volume in a 5mL volumetric flask, shaking up, and filtering with a 0.45-micrometer microporous membrane to obtain a sample solution.
Preferably, the preparation method of the control solution comprises the following steps: and (3) dissolving the ganoderic acid A reference substance into methanol to prepare a solution of 100-200 mu g/mL, thus obtaining the ganoderic acid A.
More preferably, the preparation method of the control solution is as follows: dissolving ganoderic acid A control in methanol to obtain 133 μ g/mL solution.
As a preferable scheme of the invention, the method for measuring the content of the active ingredient ganoderic acid A in the ganoderma lucidum syrup comprises the following steps:
(1) preparation of a test solution: precisely measuring 10-30 mL of ganoderma lucidum syrup, extracting with 30-60 mL of ethyl acetate, repeatedly extracting for 2-4 times, combining upper layer liquid, evaporating to dryness, dissolving residues with methanol, fixing the volume in a volumetric flask of 5-10 mL, shaking up, and filtering with a microporous membrane to obtain a test solution;
(2) preparation of control solutions: dissolving a ganoderic acid A reference substance into methanol to prepare a solution of 100-200 mug/mL, so as to obtain the ganoderic acid A;
(3) the determination method comprises the following steps: respectively and precisely sucking 5-10 mu L of each of the test solution and the mixed reference solution, injecting the solutions into a high performance liquid chromatograph, measuring and calculating;
the chromatographic conditions of the assay were: an insertstation C18 chromatography column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05 percent to 0.5 percent, and the gradient elution method is adopted; the detection wavelength is 200-400 nm, the column temperature is 35-40 ℃, and the flow rate is 0.5-2.0 mL/min.
Further preferably, the method for measuring the content of the active ingredient ganoderic acid A in the ganoderma lucidum syrup comprises the following steps:
(1) preparation of a test solution: precisely measuring 20mL of ganoderma lucidum syrup, placing the ganoderma lucidum syrup into a conical flask with a plug, extracting with 50mL of ethyl acetate, repeatedly extracting for 3 times, combining upper layer liquid, evaporating to dryness, dissolving residues with methanol, fixing the volume in a 5mL volumetric flask, shaking up, and filtering with a 0.45-micrometer microporous membrane to obtain a sample solution;
(2) preparation of control solutions: dissolving ganoderic acid A control in methanol to obtain 133 μ g/mL solution;
(3) the determination method comprises the following steps: precisely sucking 10 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, measuring, and calculating;
the chromatographic conditions of the assay were: InertSustain C18 chromatographic column with model of 250 × 4.6mm,5 μm, Shimadzu chromatographic column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.1 percent, and the gradient elution method is adopted; the detection wavelength is 258nm, the column temperature is 35 ℃, and the flow rate is 1.0 mL/min.
Through actual detection, each ganoderma lucidum syrup contains ganoderic acid A (C)30H42O7) It should be counted that the content is not less than 500. mu.g/count.
The invention realizes the following beneficial effects:
1. the invention establishes a high performance liquid chromatography method for detecting the content of the ganoderic acid A in the ganoderma lucidum syrup for the first time, and based on the invention, an effective quality detection and control method for the content of the ganoderic acid A can be established, so that the quality of the ganoderma lucidum syrup product is better improved.
2. The gradient elution method suitable for detecting the active ingredients of the ganoderma lucidum syrup is accurately summarized, the retention time of the active ingredient ganoderic acid A is shortened, the content determination time is shortened, the detection effect of the detection efficiency is stably improved, and meanwhile, the using amount of the mobile phase is reduced.
3. The method has the advantages of stable detection baseline, high separation degree, high detection accuracy and good durability.
Drawings
FIG. 1 is a chromatogram of a test solution of example 1;
FIG. 2 is a chromatogram of a control solution of example 1;
FIG. 3 is a standard curve diagram of ganoderic acid A;
FIG. 4 is a 30 ℃ control chromatogram for column temperature durability test;
FIG. 5 is a 35 ℃ control chromatogram for column temperature durability test;
FIG. 6 is a 40 ℃ control chromatogram for column temperature durability test.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise specified, all the raw materials and equipment used in this example were those conventionally available in the art.
The apparatus and reagents referred to in the following examples include: a liquid chromatograph Agilent 1260 Infinity; column InertSustain C18 (4.6X 250mm, 5 μm); electron analytical balance METTLER TOLEDO AB204-S (one hundred thousandth); methanol (chromatographically pure, miuiou chemical reagents ltd, tianjin); acetonitrile (chromatographically pure, Honeywell corporation); formic acid (analytically pure, shin-shin science and technology development ltd, Tianjin); ethyl acetate (analytical purity, hunan hui hong reagent limited); the water is the Wahaha purified water.
A ganoderic acid A control (batch No. 111985-201601, purity: 95.0%) was purchased from China food and drug testing research institute.
Example 1 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was examined as follows:
(1) preparation of a test solution: precisely measuring 20mL of ganoderma lucidum syrup, extracting with 50mL of ethyl acetate, repeatedly extracting for 3 times, combining upper layer liquid, evaporating to dryness, dissolving the residue with methanol, fixing the volume in a 5mL volumetric flask, shaking up, and filtering with a 0.45-micrometer microporous membrane to obtain a test solution;
(2) preparation of control solutions: dissolving ganoderic acid A control in methanol to obtain 133 μ g/mL solution;
(3) the determination method comprises the following steps: respectively and precisely sucking 10 μ L of each of the test solution and the reference solution, injecting into a liquid chromatograph, measuring, and calculating;
the chromatographic conditions of the assay are: InertSustain C18 chromatographic column with model of 250 × 4.6mm,5 μm, Shimadzu chromatographic column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.1 percent, and the gradient elution method is adopted; 0-5 min, 5% A; 5-7 min, 5% → 36% A; 7-30 min, 36% A; 30-31 min, 36% → 85% A; 31-45 min, 85% A; the detection wavelength is 258nm, the column temperature is 35 ℃, and the flow rate is 1.0 mL/min.
The detection is carried out according to the method of the embodiment, and the result shows that the detection base line of the test sample and the reference solution is stable, the separation degree of each target component in the test sample is good, the retention time of the target component ganoderic acid A is 25.38min, and the detection can be completed within 45 min.
Example 2 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was tested as follows:
(1) preparation of a test solution: precisely measuring 10mL of ganoderma lucidum syrup, extracting with 30mL of ethyl acetate, repeatedly extracting for 2 times, combining upper layer liquid, evaporating to dryness, dissolving the residue with methanol, fixing the volume in a 10mL volumetric flask, shaking up, and filtering with a 0.45-micrometer microporous membrane to obtain a test solution;
(2) preparation of control solutions: dissolving ganoderic acid A reference substance in methanol to obtain 100 μ g/mL solution;
(3) the determination method comprises the following steps: precisely sucking 5 μ L of each of the sample solution and the reference solution, injecting into a liquid chromatograph, measuring, and calculating;
the chromatographic conditions of the assay were the same as in example 1.
The detection is carried out according to the method of the embodiment, and the result shows that the detection base line of the test sample and the reference solution is stable, the separation degree of each target component in the test sample is good, the effect is equivalent to that of the embodiment 1, the retention time of the target component ganoderic acid A is 25.01min, and the detection can be completed within 45 min.
Example 3 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was tested as follows:
(1) preparation of a test solution: precisely measuring 30mL of ganoderma lucidum syrup, extracting with 60mL of ethyl acetate, repeatedly extracting for 4 times, combining upper layer liquid, evaporating to dryness, dissolving the residue with methanol, fixing the volume in a 5mL volumetric flask, shaking up, and filtering with a 0.45-micrometer microporous membrane to obtain a test solution;
(2) preparation of control solutions: : dissolving ganoderic acid A reference substance in methanol to obtain 200 μ g/mL solution;
(3) the determination method comprises the following steps: precisely sucking 8 μ L of each of the sample solution and the reference solution, injecting into a liquid chromatograph, measuring, and calculating;
the chromatographic conditions of the assay were the same as in example 1.
The detection is carried out according to the method of the embodiment, and the result shows that the detection base line of the test sample and the reference solution is stable, the separation degree of each target component in the test sample is good, the effect is equivalent to that of the embodiment 1, the retention time of the target component ganoderic acid A is 25.49min, and the detection can be completed within 45 min.
Example 4 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was examined as follows:
(1) preparation of a test solution: the same as example 1;
(2) preparation of control solutions: the same as example 1;
(3) the determination method comprises the following steps: respectively and precisely sucking 10 μ L of each of the test solution and the reference solution, injecting into a liquid chromatograph, measuring, and calculating;
the chromatographic conditions of the assay are: InertSustain C18 chromatographic column with model of 250 × 4.6mm,5 μm, Shimadzu chromatographic column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05 percent, and the gradient elution method is adopted; 0-5 min, 2% A; 5-7 min, 2% → 30% A; 7-30 min, 30% A; 30-31 min, 30% → 70% A; 31-45 min, 70% A; the detection wavelength is 200nm, the column temperature is 40 ℃, and the flow rate is 0.5 mL/min.
The detection according to the method of the embodiment shows that the detection base line of the test sample and the reference solution is stable, the separation degree of each target component in the test sample is good, the effect is slightly worse than that of the embodiments 1 to 3, the retention time of the target component ganoderic acid A is 26.18min, and the detection can be completed within 47 min.
Example 5 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was examined as follows:
(1) preparation of a test solution: the same as example 1;
(2) preparation of control solutions: the same as example 1;
(3) the determination method comprises the following steps: respectively and precisely sucking 10 μ L of each of the test solution and the mixed reference solution, injecting into a liquid chromatograph, measuring, and calculating;
the chromatographic conditions of the assay are: InertSustain C18 chromatographic column with model of 250 × 4.6mm,5 μm, Shimadzu chromatographic column; the mobile phase A is acetonitrile, the mobile phase B is a formic acid water solution with the volume fraction of 0.5%, and the gradient elution method is 0-5 min and is 10% of A; 5-7 min, 10% → 50% A; 7-30 min, 50% A; 30-31 min, 50% → 90% A; 31-45 min, 90% A; the detection wavelength is 400nm, the column temperature is 35 ℃, and the flow rate is 2.0 mL/min.
The detection according to the method of the embodiment shows that the detection base line of the test sample and the reference solution is stable, the separation degree of each target component in the test sample is good, the effect is equivalent to that of the embodiment 4, the effect is slightly worse than that of the embodiments 1 to 3, the retention time of the target component ganoderic acid A is 26.22min, and the detection can be completed within 47 min.
Example 6 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was tested as follows:
(1) preparation of a test solution: the same as example 1;
(2) preparation of control solutions: the same as example 1;
(3) the determination method comprises the following steps: respectively and precisely sucking 10 μ L of each of the test solution and the mixed reference solution, injecting into a liquid chromatograph, measuring, and calculating;
the chromatographic conditions of the assay are: InertSustain C18 chromatographic column with model of 250 × 4.6mm,5 μm, Shimadzu chromatographic column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.5 percent, and the gradient elution method is adopted; 0-5 min, 8% A; 5-7 min, 8% → 40% A; 7-30 min, 40% A; 30-31 min, 40% → 80% A; 31-45 min, 80% A; the detection wavelength is 400nm, the column temperature is 35 ℃, and the flow rate is 2.0 mL/min.
The detection according to the method of the embodiment shows that the detection base line of the test sample and the reference solution is stable, the separation degree of each target component in the test sample is good, the effect is equivalent to that of the embodiment 4 and the embodiment 5, the effect is slightly worse than that of the embodiment 1 to the embodiment 3, the retention time of the target component ganoderic acid A is 26.25min, and the detection time can be completed within 47 min.
Comparative example 1 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was examined as follows:
compared to example 1, except that the chromatographic conditions were a C18 column, 250mm 4.6mm (i.d.),5 μm, or equivalent size column; mobile phase: acetonitrile-0.1% aqueous formic acid 30: 70; flow rate 1mL/min, column temperature: at 40 ℃, the wavelength is 258nm, and the sample injection amount is 20 mu L; the rest is the same as in example 1.
The same batch of samples as in example 1 were respectively measured by the method in the comparative example, and the results show that the retention time of the target component ganoderic acid A is 28.89min, 50min is required for complete detection, the degree of separation between the target component ganoderic acid A and other components is poor, and the degree of separation effect is inferior to that of examples 1-6 when the chromatographic conditions in the comparative example are used for measurement.
Comparative example 2 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was examined according to the following method:
compared with example 1, the difference is that the chromatographic conditions are a chromatographic column ZORBAX SB-C18(150 mm. times.4.6 mm,5 μm); mobile phase: 1% acetic acid solution: methanol is 40%: 60 percent; flow rate 0.5mL/min, column temperature: at 25 ℃, the wavelength is 254nm, and the sample injection amount is 5 mu L; the rest is the same as in example 1.
The same batch of samples as in example 1 were respectively measured by the above methods in this comparative example, and the results show that the retention time of the target component ganoderic acid a was 13.619min, and 20min was required for complete measurement, but the separation degree between the target component ganoderic acid a and other components was poor, the baseline of measurement was not stable, and content data could not be accurately calculated.
Comparative example 3 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was tested according to the following method:
compared with example 1, the difference is that the chromatographic conditions are Kromasil 100-5C18 chromatography (250 mm. times.4.6 mm,5 μm); the mobile phase is acetonitrile-0.1% formic acid solution; gradient elution is adopted, and gradient elution conditions are adopted; 0-5 min, 27% -30% acetonitrile: 5-50 min, 30% acetonitrile: 50-60 min, 30-35% acetonitrile; the column temperature was 25 ℃; flow rate: 0.8 mL/min; the sample volume is 10 mu L; the detection wavelength is 254 nm; the rest is the same as in example 1.
The same batch of samples as in example 1 were respectively measured by the above methods in this comparative example, and the results show that the measurement performed under the chromatographic conditions described in this comparative example does not distinguish the target component ganoderic acid a from other impurity components well, has a low degree of separation, cannot accurately measure the content of the target component ganoderic acid a, and requires 60min for complete measurement.
Comparative example 4 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was examined according to the following method:
compared with the example 1, the difference is that the chromatographic conditions are that a chromatographic column is Promosil-C18(250mm multiplied by 4.6mm,5 mu m), a mobile phase is acetonitrile, namely a formic acid aqueous solution with a volume fraction of 0.03% (0-35 min, the volume fraction of acetonitrile is increased from 25% to 35%; 35-55 min, the volume fraction of acetonitrile is increased from 35% to 45%; 55-65 min, the volume fraction of acetonitrile is increased from 45% to 55%), a flow rate is 0.8mL/min, a detection wavelength is 254nm, and a sample introduction amount is 20 mu L; the rest is the same as in example 1.
The comparative example respectively measures the same batch of samples as in example 1 by adopting the method, and the result shows that when the chromatographic conditions are used for measurement, the detection baseline is not stable, the target component ganoderic acid A and other impurity components cannot be well distinguished, the separation degree is low, the content of the target component ganoderic acid A cannot be accurately measured, and 60min is required for complete detection.
Comparative example 5 the content of the active ingredient ganoderic acid a in the ganoderma lucidum syrup was examined as follows:
the difference compared to example 1 is that the chromatographic conditions are: an Altima C18 chromatography column (4.6 mm. times.150 mm,5 μm); the mobile phase is acetonitrile-0.04% formic acid solution; gradient elution conditions: 0-10 min, 20-25% acetonitrile; 10-20 min, 25% -30% acetonitrile; 20-50 min, 30% acetonitrile; 50-65 min, 30-38% acetonitrile; 65-80 min, 38% acetonitrile; the column temperature is 15 ℃; the flow rate is 1.0 mL/min; the sample volume is 20 mu L; the detection wavelength is 254 nm; the rest is the same as in example 1.
The results show that the detection by the method of the comparative example has unstable spectrogram base line, the detection time is 60 minutes after the peak emergence time of the target component ganoderic acid A is 30 minutes, and in the detection method, the separation degree of the ganoderic acid A and impurity components is poor, so that the content of the ganoderic acid A cannot be stably detected.
Example 7 methodological investigation and assay tests:
the sample solution and the control solution were treated in the same manner as in example 1, and the following tests were carried out under the same chromatographic conditions as in example 1:
(1) specificity test: sampling 10 μ L of reference solution and sample solution, respectively, recording chromatogram, and showing that the relative retention time of ganoderic acid A reference is 25.38min, the separation degree of ganoderic acid A in sample solution is 3.03, and the theoretical plate number is 29908.
(2) And (3) linear relation investigation: precisely weighing 0.0112g of ganoderic acid A reference substance, placing into a 10mL measuring flask, adding a proper amount of methanol for ultrasonic treatment to dissolve, taking out, cooling, adding methanol for diluting to a scale, and shaking up to obtain 1064 mu g/mL of ganoderic acid A reference substance storage solution; precisely diluting the stock solution of ganoderic acid A into control solutions with concentrations of 8.3125 μ g/mL, 16.625 μ g/mL, 33.25 μ g/mL, 66.5 μ g/mL, 133 μ g/mL, 266 μ g/mL and 532 μ g/mL respectively, shaking, precisely sucking 10 μ L of the stock solution together with the control solutions respectively, injecting into a liquid chromatograph, and measuring by high performance liquid chromatography (0512 in the fourth division of the national pharmacopoeia 2015). A standard curve was plotted with the concentration (. mu.g/mL) as the abscissa and the peak area as the ordinate. See table 1. The regression equation: Y8.6338X + 13.4785; correlation coefficient: r is 1.0000.
The results show that the ganoderic acid A has good linearity in the range of 8.3125-1064. mu.g/mL.
TABLE 1 Linearic acid A Linear relationship (n ═ 3)
(2) And (3) testing the precision of an instrument: 20mL of ganoderma lucidum syrup (batch number: 20170401) is precisely measured, and the operation is carried out according to the text item of preparation and determination method of the test solution, the sample is continuously fed for 6 times, and the determination result is shown in Table 2.
Table 2 instrument precision test results (n ═ 6)
The results show that the instrument precision is good.
(3) And (3) stability test: 20mL of this product (lot: 20170401) was precisely measured, and the test results are shown in Table 3, following the preparation and measurement of the test solution in example 1, after the preparation, the sample was injected for 0, 2, 4, 8, 12, and 24 hours, respectively.
Table 3 stability test results (n ═ 6)
The results show that the test solution has good peak area stability within 24 hours.
(4) And (3) repeatability test: 20mL (6 parts in total) of this product (lot No. 20170401) was precisely measured and subjected to the procedures described in the preparation and measurement methods of the test solution described in example 1. The results are shown in Table 4.
Table 4 results of repetitive tests (n ═ 6)
The result shows that RSD is less than 3.0%, which indicates that the repeatability of the method is good.
(5) Intermediate precision test: 20mL (total 6 parts) of this product (lot: 20170401) was precisely measured, and the content of this product was measured by two different analysts using different instruments according to the preparation of the test solution under example 1. The results are shown in Table 5.
TABLE 5 intermediate precision test results (n-12)
The results show that the method has good intermediate precision.
(6) Sample recovery rate test: precisely measuring 20mL (total 6 parts) of the product (lot number: 20170401) by a sample-adding recovery method, separately placing into conical flasks with stoppers, precisely measuring 14.0mg of ganoderic acid A (purity 95%) as a reference substance, placing into 10mL measuring flasks, adding methanol to dissolve and dilute to scale, shaking up, precisely measuring 5mL from the middle into 10mL measuring flasks, adding methanol to dissolve and dilute to scale, shaking up to prepare a 665 mu g/mL reference substance solution, precisely sucking 1mL (total 6 parts), placing into the conical flasks, extracting with 50mL ethyl acetate, repeatedly extracting for 3 times, taking the supernatant, evaporating to dryness, dissolving the residue with methanol, fixing the volume in 10mL measuring flask, shaking up, filtering with 0.45 mu m microporous membrane, precisely sucking 10 mu L of subsequent filtrate, injecting into a liquid chromatograph, and measuring to obtain the product. The results are shown in Table 6. The content of ganoderic acid A in the ganoderma lucidum syrup (batch No. 20170401) was 29.51. mu.g/mL.
TABLE 6 recovery test results for ganoderic acid A samples (n ═ 6)
(7) Durability tests were conducted to examine the durability of acids of different chromatographic columns, different column temperatures, different flow rates, different pH to the conditions of the spectrum, and the results are shown in table 7.
TABLE 7 durability test results for ganoderic acid A
The result shows that the determination results of different chromatographic columns and different flow rates and pH values under acid conditions are basically consistent, the RSD percent is less than 4 percent, and the peak type of the ganoderic acid A in the chromatogram is sharp and symmetrical, and the separation degree is good; the results measured under different column temperatures are inconsistent, and the peak shape of the ganoderic acid A measured at 30 ℃ is asymmetric. The method shows that the acid with different chromatographic columns, different flow rates and different pH has good durability, and the temperature of the measured column needs to be controlled to be 35 ℃.
The research result experiments show that the method for measuring the content of the ganoderic acid A in the ganoderma lucidum syrup has strong specificity, the stability, the repeatability, the intermediate precision, the sample adding recovery rate and the like all accord with the regulations, and the durability of the chromatographic condition is better, so that the liquid chromatographic condition can be used for measuring the content of the ganoderic acid A in the ganoderma lucidum syrup.
(8) And (3) sample content determination: two samples to be tested were precisely measured, and the content of ganoderic acid A in 10 samples was measured in accordance with the procedure for preparing the test solution and the method for measuring in example 1, and the results are shown in Table 8.
TABLE 8 eleven batches of Ganoderic acid A assay results (n ═ 2)
The content of ganoderic acid A in ten samples is 677.64 μ g/piece, and each piece contains ganoderic acid A (C)30H42O7) It should not be less than 500. mu.g.
From the experimental results, the content of the ganoderic acid A in the ganoderma lucidum syrup is measured under the condition of the invention, the time and the solvent are saved, better peak type and theoretical plate number are obtained, the target component and the impurities are completely separated, the separation degree is good, the specificity is strong, the durability is good, and the quantitative analysis method established by the experiment is the optimal content detection method for the ganoderma lucidum syrup.
The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are intended to illustrate the principles of the invention, and that various changes and modifications, which will be apparent to those skilled in the art, may be made without departing from the spirit and scope of the invention and fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (11)
1. The method for measuring the content of the ganoderic acid A in the ganoderma lucidum syrup is characterized by comprising the following steps: the method comprises the following steps: preparing a test solution, preparing a reference solution and measuring by high performance liquid chromatography;
the chromatographic conditions measured by the high performance liquid chromatography are as follows: an insertstation C18 chromatography column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05 percent to 0.5 percent, and the gradient elution method is adopted; the detection wavelength is 258nm, the column temperature is 35 ℃, and the flow rate is 0.5-2.0 mL/min;
the gradient elution method comprises the following steps: 0-5 min, 2% -10% A; 5-7 min, 2% -10% → 30% -50% A; 7-30 min, 30% -50% A; 30-31 min, 30% -50% → 70% -90% A; 31-45 min, 70% -90% A.
2. The method for determining the content of the ganoderic acid A in the ganoderma lucidum syrup according to claim 1, wherein the method comprises the following steps: the InertSustain C18 chromatographic column has a model of 250 × 4.6mm,5 μm, Shimadzu chromatographic column.
3. The method for determining the content of the ganoderic acid A in the ganoderma lucidum syrup according to claim 1, wherein the method comprises the following steps: the mobile phase B is a formic acid aqueous solution with the volume fraction of 0.1%, and the flow rate is 1.0 mL/min.
4. The method for determining the content of the ganoderic acid A in the ganoderma lucidum syrup according to claim 1, wherein the method comprises the following steps: the gradient elution method comprises the following steps: 0-5 min, 5% A; 5-7 min, 5% → 36% A; 7-30 min, 36% A; 30-31 min, 36% → 85% A; 31-45 min, 85% A.
5. The method for determining the content of the ganoderic acid A in the ganoderma lucidum syrup according to claim 1, wherein the method comprises the following steps: the preparation method of the test solution comprises the following steps: precisely measuring 10-30 mL of ganoderma lucidum syrup, extracting with 30-60 mL of ethyl acetate, repeatedly extracting for 2-4 times, combining upper layer liquid, evaporating to dryness, dissolving residues with methanol, fixing the volume in a 5-10 mL volumetric flask, shaking up, and filtering with a microporous membrane to obtain a sample solution.
6. The method for determining the content of the ganoderic acid A component in the ganoderma lucidum syrup according to claim 5, wherein the method comprises the following steps: the preparation method of the test solution comprises the following steps: precisely measuring 20mL of ganoderma lucidum syrup, extracting with 50mL of ethyl acetate, repeatedly extracting for 3 times, combining upper layer liquid, evaporating to dryness, dissolving the residue with methanol, fixing the volume in a 5mL volumetric flask, shaking up, and filtering with a 0.45-micrometer microporous membrane to obtain a test solution.
7. The method for determining the content of the ganoderic acid A in the ganoderma lucidum syrup according to claim 1, wherein the method comprises the following steps: the preparation method of the reference substance solution comprises the following steps: and (3) dissolving the ganoderic acid A reference substance into methanol to prepare a solution of 100-200 mu g/mL, thus obtaining the ganoderic acid A.
8. The method for determining the content of the ganoderic acid A component in the ganoderma lucidum syrup according to claim 7, wherein the method comprises the following steps: the preparation method of the reference substance solution comprises the following steps: dissolving ganoderic acid A control in methanol to obtain 133 μ g/mL solution.
9. The method for determining the content of the ganoderic acid A in the ganoderma lucidum syrup according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) preparation of a test solution: precisely measuring 10-30 mL of ganoderma lucidum syrup, extracting with 30-60 mL of ethyl acetate, repeatedly extracting for 2-4 times, combining upper layer liquid, evaporating to dryness, dissolving residues with methanol, fixing the volume in a volumetric flask of 5-10 mL, shaking up, and filtering with a microporous membrane to obtain a test solution;
(2) preparation of control solutions: dissolving a ganoderic acid A reference substance into methanol to prepare a solution of 100-200 mug/mL, so as to obtain the ganoderic acid A;
(3) the determination method comprises the following steps: precisely sucking 5-10 μ l of each of the test solution and the mixed reference solution, injecting into a liquid chromatograph, measuring, and calculating to obtain the final product;
the chromatographic conditions of the assay were: an insertstation C18 chromatography column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05 percent to 0.5 percent, and the gradient elution method is adopted; the flow rate is 0.5-2.0 mL/min.
10. The method for determining the content of the ganoderic acid A component in the ganoderma lucidum syrup according to claim 9, wherein the method comprises the following steps: the method comprises the following steps:
(1) preparation of a test solution: precisely measuring 20mL of ganoderma lucidum syrup, extracting with 50mL of ethyl acetate, repeatedly extracting for 3 times, combining upper layer liquid, evaporating to dryness, dissolving the residue with methanol, fixing the volume in a 5mL volumetric flask, shaking up, and filtering with a 0.45-micrometer microporous membrane to obtain a test solution;
(2) preparation of control solutions: dissolving ganoderic acid A control in methanol to obtain 133 μ g/mL solution;
(3) the determination method comprises the following steps: respectively and precisely sucking 10 μ L of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating;
the chromatographic conditions of the assay were: InertSustain C18 chromatographic column with model of 250 × 4.6mm,5 μm, Shimadzu chromatographic column; the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.1 percent, and the gradient elution method is adopted; the detection wavelength is 258nm, the column temperature is 35 ℃, and the flow rate is 1.0 mL/min.
11. The method for determining content of ganoderic acid A in ganoderma lucidum syrup according to any one of claims 1 to 10, wherein ganoderic acid A (C) is contained in each ganoderma lucidum-containing syrup30H42O7) It should be counted that the content is not less than 500. mu.g/count.
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