CN113311085B - Method for measuring content of chlorogenic acid and linarin in wild chrysanthemum granules - Google Patents
Method for measuring content of chlorogenic acid and linarin in wild chrysanthemum granules Download PDFInfo
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Abstract
The invention provides a method for measuring the content of chlorogenic acid and linarin in wild chrysanthemum granules, which relates to the technical field of quality detection, and comprises the following steps: (1) preparation of a reference solution: dissolving chlorogenic acid reference substance and linarin reference substance with solvent respectively, and metering volume to obtain the final product; (2) sample solution preparation: grinding flos Chrysanthemi Indici granule, pretreating, adding solvent, heating under reflux, shaking, standing and filtering; (3) determination of chromatographic conditions: the mobile phase is acetonitrile and phosphoric acid aqueous solution; (4) and (3) determination: and (4) injecting the sample solution and the reference substance solution into a liquid chromatograph for determination to obtain the product.
Description
Technical Field
The invention relates to the technical field of quality detection, and particularly relates to a method for determining the content of chlorogenic acid and linarin in wild chrysanthemum flower granules.
Background
Flos Chrysanthemi Indici is the dried head-like inflorescence of Chrysanthemum indicum L.of Compositae. Collected in the beginning of blooming in autumn and winter, and dried in the sun or steamed and dried in the sun. Bitter and pungent in flavor and slightly cold in nature. It enters liver and heart meridians. Can be used for clearing away heat and toxic materials, purging pathogenic fire, and calming liver. Can be used for treating furuncle, carbuncle, swelling, conjunctival congestion, swelling and pain, headache, and vertigo. Wild chrysanthemum has more than 2000 years of traditional medicine history in China and is widely used for treating herpes, swelling and lymphodynia. To date, 190 chemical components have been isolated and identified from this plant, including flavonoids, terpenoids, phenylpropanoids, and phenolic acids, among others. A large number of researches show that the wild chrysanthemum extract has various pharmacological activities of resisting inflammation, resisting oxidation, resisting bacteria, resisting cancer, regulating immunity, protecting liver and the like. At present, various traditional Chinese medicine preparations taking wild chrysanthemum as raw materials are available, such as wild chrysanthemum suppositories, wild chrysanthemum granules and the like.
At present, the content of linarin is usually detected by a method under the item of flos chrysanthemi indici or flos chrysanthemi indici suppository in 'Chinese pharmacopoeia' 2020 edition, but certain waste is caused while the integral quality evaluation cannot be met by only detecting a single component. To solve the problems, chinese patent CN106841478A discloses a method for simultaneously determining the content of seven components in wild chrysanthemum, which specifically comprises: 1) preparing a reference stock solution; 2) preparing a test solution; 3) respectively injecting the reference substance solution and the test solution into a chromatograph for detection, wherein the chromatographic column is Syncronis C18, and the mobile phase is acetonitrile-0.5% phosphoric acid aqueous solution for gradient elution; the flow rate is 0.5mL min < -1 >, the detection wavelength is 334nm, the column temperature is 35 ℃, and the sample injection amount is 2 mu L; 4) sampling the mixed reference substance solution at 0.2, 0.5, 1, 2, 3, 4 and 5 μ L, respectively, measuring peak area under the chromatographic conditions, and drawing a standard curve by taking the sampling amount of each component as a horizontal coordinate and the peak area as a vertical coordinate to obtain a regression equation and a correlation coefficient. The method can detect the contents of various components simultaneously, but the method has relatively low detection precision for the linarin and the chlorogenic acid, and particularly has poor overall results such as stability, sample adding recovery rate and the like for the chlorogenic acid. Chinese patent CN110579549A discloses a quality detection method of wild chrysanthemum flower formula particles and application thereof, the method comprises the steps of constructing a fingerprint of the wild chrysanthemum flower formula particles, establishing a reference fingerprint, analyzing the similarity of wild chrysanthemum flower formula particles in different batches, evaluating the quality consistency of the wild chrysanthemum flower formula particles in different manufacturers and the same manufacturer in different batches by means of a principal component analysis and cluster analysis mode identification method, and determining the content of 10 chromatographic peaks in the fingerprint of the wild chrysanthemum flower formula particles by combining a multi-evaluation method. However, the method also has the problem of low detection accuracy, and meanwhile, the stability of the detection for the chlorogenic acid is slightly poor, the detection is mainly performed for the high content of the chlorogenic acid, and the detection is difficult when the content of the chlorogenic acid in the particles is low. However, Chinese patent CN102928523B has the problem of low precision, and it is difficult to accurately detect linarin.
Aiming at the problems of low detection precision and stability of chlorogenic acid or linarin in wild chrysanthemum flower granules and difficulty in detecting chlorogenic acid or linarin in the prior art, the establishment of the content determination method of linarin in wild chrysanthemum flower granules is very key for controlling the product quality and improving the drug standard.
Disclosure of Invention
The invention provides a method for measuring the content of chlorogenic acid and linarin in wild chrysanthemum granules aiming at the problems in the prior art,
in order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for measuring the content of chlorogenic acid and linarin comprises the following steps:
(1) preparation of a reference solution: dissolving chlorogenic acid reference substance and linarin reference substance with solvent respectively, and metering volume to obtain the final product;
(2) preparation of a sample solution: grinding flos Chrysanthemi Indici granule, pretreating, adding solvent, heating under reflux, shaking, standing and filtering;
(3) determination of chromatographic conditions: the mobile phase is acetonitrile and phosphoric acid aqueous solution;
(4) and (3) determination: and (4) carrying out liquid chromatography determination on the sample solution and the reference solution to obtain the product.
Further, the solvent in the step (1) is a methanol solution, and the mass fraction of the methanol solution is 99.5%.
Further, the concentration of the chlorogenic acid reference substance in the step (1) is 30-155 mu g/mL, and the concentration of the linarin reference substance is 12.3-62 mu g/mL.
Further, the pretreatment in the step (2) specifically comprises the steps of soaking the ground wild chrysanthemum flower particles in a mixed solution of ethanol and ether, drying, cooling, heating for the second time, and cooling to the normal temperature to obtain the wild chrysanthemum flower tea.
Further, the volume ratio of the ethanol to the diethyl ether is 4-5: 1.
Further, the second heating specifically refers to the first heating to 30-40 ℃ and the second heating to 45-50 ℃.
Further, the concentration of the sample solution in the step (2) is 30-90 mg/mL.
Further, the liquid chromatography assay in step (4) specifically includes gradient elution, and the elution method includes:
0-10min, wherein the volume of the acetonitrile accounts for 5% -15% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 95% -85% of the total volume of the mobile phase;
10-15min, wherein the volume of the acetonitrile is increased from 5% -15% of the total volume to 20% -35%, and the volume of the acetonitrile is reduced from 95% -85% of the total volume to 80% -65%;
15-25min, wherein the volume of the acetonitrile accounts for 20-35% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 80-65% of the total volume of the mobile phase;
25-30min, wherein the volume of the acetonitrile is reduced from 20% -35% of the total volume to 5% -15%, and the volume of the acetonitrile is increased from 80% -65% of the total volume to 95% -85%;
and (3) 30-40min, wherein the volume of the acetonitrile accounts for 5-15% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 95-85% of the total volume of the mobile phase.
Preferably, the elution method comprises:
0-10min, wherein the volume of the acetonitrile accounts for 13% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 87% of the total volume of the mobile phase;
10-15min, increasing the volume of acetonitrile from 13% of the total volume to 30%, and reducing the volume of acetonitrile from 87% of the total volume to 70%;
15-25min, wherein the volume of the acetonitrile accounts for 30% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 70% of the total volume of the mobile phase;
25-30min, the volume of the acetonitrile is reduced from 30% to 13% of the total volume, and the volume of the acetonitrile is increased from 70% to 87% of the total volume;
30-40min, wherein the volume of the acetonitrile accounts for 13% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 87% of the total volume of the mobile phase.
Further, the theoretical plate number of chlorogenic acid in the chromatographic conditions of the liquid chromatography determination in the step (4) is not less than 12000, and the theoretical plate number of linarin is not less than 140000.
Further, the chromatographic conditions of the liquid chromatography assay are: kromasil 100-5C 18250X 4.6mm SN: e103500; ultraviolet detection wavelength: 334 nm; column temperature: at 32 ℃; sample injection volume: 10 μ L.
Further, the preparation method of the wild chrysanthemum flower particles comprises the following steps: taking 333g of wild chrysanthemum, adding 10 times of pure water, decocting for 1 hour, filtering, adding 5 times of pure water for the third time, decocting for half an hour, filtering, combining filtrates, distilling the filtrate at 80 ℃ under reduced pressure until the relative density reaches 1.16, cooling the clear paste to room temperature, adding 95% ethanol until the alcohol content reaches 60%, standing for 24 hours, filtering, distilling the filtrate under reduced pressure to recover the ethanol, concentrating until the relative density reaches 1.26(80 ℃), cooling, adding 960g of sucrose powder, granulating by a wet granulation method to prepare granules, and drying at 60 ℃ for 0.5 hour. The wild chrysanthemum granules are prepared according to the preparation method of the wild chrysanthemum granules in the national drug standards, and the examination is in accordance with the requirements.
The technical effects obtained by the invention are as follows:
the method for measuring the content of chlorogenic acid and linarin in the wild chrysanthemum flower particles by using the HPLC method is simple, convenient, rapid, accurate and good in repeatability, can be used for controlling and detecting the product quality of the wild chrysanthemum flower particles, and can be used as a content measuring method of the wild chrysanthemum flower particles.
Drawings
FIG. 1 is a chromatogram of the sample solution, blank control solution, chlorogenic acid and linarin control solution of example 3.
Wherein, 1 in the reference signs is a sample solution, 2 is a chlorogenic acid and linarin control solution, and 3 is a blank control solution.
Detailed Description
It should be noted that the liquid chromatograph used in the present invention is a type LC-20A high performance liquid chromatograph manufactured by shimadzu corporation, japan, and the methanol used in the present invention is analytically pure and purchased from mitsui chemical ltd, tianjin; acetonitrile was chromatographically pure, purchased from merck, germany; the chlorogenic acid reference substance is provided by China food and drug verification institute, the batch number is 110753-containing 201817, and the purity is 96.8%; the linarin control was provided by the institute of food and drug assay, lot number 111528-. The other raw materials are common commercial products, and thus the sources thereof are not particularly limited.
Example 1
A method for measuring the content of chlorogenic acid and linarin comprises the following steps:
(1) preparation of a reference solution: dissolving 15.00mg of a chlorogenic acid reference substance and 6.15mg of a linarin reference substance respectively with a solvent, and metering to a 100mL volumetric flask to obtain the chlorogenic acid reference substance, wherein the concentration of the chlorogenic acid reference substance is 150 mu g/mL, and the concentration of the linarin reference substance is 61.5 mu g/mL;
(2) preparation of a sample solution: taking 0.25g of wild chrysanthemum particles, grinding, placing the wild chrysanthemum particles into a mixed solution of ethanol and ether (prepared by using 70% by mass of ethanol solution and 30% by mass of ether solution) with a volume ratio of 4:1, soaking, drying, cooling, heating to 30 ℃ for the first time, heating to 45 ℃ for the second time, cooling to normal temperature, adding 100mL of methanol, weighing, heating and refluxing for 3 hours, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the wild chrysanthemum particle;
(3) determination of chromatographic conditions: the mobile phase is acetonitrile and phosphoric acid aqueous solution; the chromatographic conditions of the liquid chromatographic determination are as follows: kromasil 100-5C 18250X 4.6mm SN: e103500; ultraviolet detection wavelength: 334 nm; column temperature: at 32 ℃; sample introduction volume: 10 mu L of the solution; the mobile phase is acetonitrile and phosphoric acid water solution with the mass fraction of 0.4 percent;
(4) and (3) determination: and (4) carrying out liquid chromatography determination on the sample solution and the reference solution to obtain the product. The liquid chromatography determination specifically comprises gradient elution, and the elution method comprises the following steps:
0-10min, wherein the volume of the acetonitrile accounts for 5% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 95% of the total volume of the mobile phase;
10-15min, increasing the volume of acetonitrile from 5% to 20% of the total volume, and reducing the volume of acetonitrile from 95% to 80% of the total volume;
15-25min, wherein the volume of the acetonitrile accounts for 20% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 80% of the total volume of the mobile phase;
25-30min, the volume of acetonitrile is reduced from 20% to 5% of the total volume, and the volume of acetonitrile is increased from 70% to 95% of the total volume;
30-40min, wherein the volume of the acetonitrile accounts for 5% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 95% of the total volume of the mobile phase.
The theoretical plate number of chlorogenic acid is not less than 12000, and the theoretical plate number of linarin is not less than 140000.
Example 2
(1) Preparation of a reference solution: dissolving 15.00mg of a chlorogenic acid reference substance and 6.15mg of a linarin reference substance respectively with a solvent, and metering to a 100mL volumetric flask to obtain the final product, wherein the concentration of the chlorogenic acid reference substance is 150 mug/mL, and the concentration of the linarin reference substance is 61.5 mug/mL;
(2) preparation of a sample solution: taking 0.25g of wild chrysanthemum granules, grinding, placing the ground wild chrysanthemum granules into a mixed solution of ethanol and ether (prepared by using 70% of ethanol solution and 30% of ether solution) in a volume ratio of 4.5:1, drying, cooling, heating to 40 ℃ for the first time, heating to 50 ℃ for the second time, cooling to normal temperature, adding 100mL of methanol, weighing, heating and refluxing for 3 hours, cooling, weighing again, complementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the wild chrysanthemum granules;
(3) determination of chromatographic conditions: the mobile phase is acetonitrile and phosphoric acid aqueous solution; the chromatographic conditions of the liquid chromatographic determination are as follows: kromasil 100-5C 18250X 4.6mm SN: e103500; ultraviolet detection wavelength: 334 nm; column temperature: at 32 ℃; sample injection volume: 10 mu L of the solution; the mobile phase is acetonitrile and phosphoric acid water solution with the mass fraction of 0.4 percent;
(4) and (3) determination: and (4) carrying out liquid chromatography determination on the sample solution and the reference solution to obtain the product. The liquid chromatography determination specifically comprises gradient elution, and the elution method comprises the following steps:
0-10min, wherein the volume of the acetonitrile accounts for 15% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 85% of the total volume of the mobile phase;
10-15min, increasing the volume of acetonitrile from 15% to 35% of the total volume, and reducing the volume of acetonitrile from 85% to 65% of the total volume;
15-25min, wherein the volume of the acetonitrile accounts for 35% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 65% of the total volume of the mobile phase;
25-30min, the volume of acetonitrile is reduced from 35% to 15% of the total volume, and the volume of acetonitrile is increased from 65% to 85% of the total volume;
30-40min, wherein the volume of the acetonitrile accounts for 15% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 85% of the total volume of the mobile phase.
The theoretical plate number of chlorogenic acid is not less than 12000, and the theoretical plate number of linarin is not less than 140000.
Example 3
(1) Preparation of a reference solution: dissolving 15.00mg of a chlorogenic acid reference substance and 6.15mg of a linarin reference substance respectively with a solvent, and metering to a 100mL volumetric flask to obtain the chlorogenic acid reference substance, wherein the concentration of the chlorogenic acid reference substance is 150 mu g/mL, and the concentration of the linarin reference substance is 61.5 mu g/mL;
(2) preparation of a sample solution: taking 0.25g of wild chrysanthemum particles, grinding, placing the wild chrysanthemum particles into a mixed solution of ethanol and ether (prepared by using 70% by mass of ethanol solution and 30% by mass of ether solution) with a volume ratio of 5:1, soaking, drying, cooling, heating to 35 ℃ for the first time, heating to 48 ℃ for the second time, cooling to normal temperature, adding 100mL of methanol, weighing, heating and refluxing for 3 hours, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the wild chrysanthemum particle;
(3) determination of chromatographic conditions: the mobile phase is acetonitrile and phosphoric acid aqueous solution; the chromatographic conditions of the liquid chromatographic determination are as follows: kromasil 100-5C 18250X 4.6mm SN: e103500; ultraviolet detection wavelength: 334 nm; column temperature: at 32 ℃; sample introduction volume: 10 mu L of the solution; the mobile phase is acetonitrile and phosphoric acid water solution with the mass fraction of 0.4 percent;
(4) and (3) determination: and (4) carrying out liquid chromatography determination on the sample solution and the reference solution to obtain the product. The liquid chromatography determination specifically comprises gradient elution, and the elution method comprises the following steps:
0-10min, wherein the volume of the acetonitrile accounts for 13% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 87% of the total volume of the mobile phase;
10-15min, increasing the volume of acetonitrile from 13% of the total volume to 30%, and reducing the volume of acetonitrile from 87% of the total volume to 70%;
15-25min, wherein the volume of the acetonitrile accounts for 30% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 70% of the total volume of the mobile phase;
25-30min, the volume of the acetonitrile is reduced from 30% to 13% of the total volume, and the volume of the acetonitrile is increased from 70% to 87% of the total volume;
30-40min, wherein the volume of the acetonitrile accounts for 13% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 87% of the total volume of the mobile phase.
The theoretical plate number of chlorogenic acid is not less than 12000, and the theoretical plate number of linarin is not less than 140000.
Comparative example 1
The only difference from example 1 is that the mobile phase of the column in step (3) was changed to acetonitrile and aqueous phosphoric acid (mass fraction of 0.4%) in a volume ratio of 13:87, the total volume of which was consistent with example 1, i.e. no gradient elution was performed.
Comparative example 2
The only difference from example 1 is that the sample in step (2) was not pretreated.
Comparative example 3
The only difference from example 1 is that the secondary heating is not performed in the pretreatment process in step (2).
Comparative example 4
The only difference from example 1 is that in the pretreatment process in step (2), no ether was added, and only an equal volume of ethanol was added.
First, precision test
According to the determination method of each example, 10. mu.L of chlorogenic acid 60. mu.g/mL and linarin 24.6. mu.g/mL were precisely pipetted, and the peak area was measured 6 times to obtain tables 1-2.
TABLE 1 precision test results (chlorogenic acid)
TABLE 2 precision test results (Menghuagan)
As can be seen from tables 1-2, the precision test results of the test methods of the examples of the present invention are significantly better than the respective ratios.
Second, stability test
Taking wild chrysanthemum particle samples, according to the method of each example, measuring peak areas at 0, 12 and 24 hours respectively, and carrying out parallel experiments for 3 parts, wherein the results are shown in tables 2-3.
TABLE 3 stability test results (chlorogenic acid)
TABLE 4 stability test results (linarin)
As can be seen from tables 3-4, the stability test results of the test methods of the examples of the present invention are clearly superior to the comparative examples.
Three, other tests
In addition, the invention also develops linear relation investigation, specificity test, sample adding recovery rate test and sample determination aiming at the embodiment 3.
(1) Investigation of linear relationships
Precisely sucking 2, 4, 6, 8 and 10mL of chlorogenic acid reference solution with the concentration of 150 mug/mL respectively, placing the chlorogenic acid reference solution in a 10mL volumetric flask respectively, fixing the volume of chromatographic methanol to the scale, and shaking up. Precisely sucking 2, 4, 6, 8 and 10mL of the linarin control solution with the concentration of 61.5 mu g/mL respectively, placing the solution in a 10mL volumetric flask respectively, fixing the volume to the scale by using chromatographic methanol, and shaking up. The sample was taken for measurement under the chromatographic conditions in example 3. And drawing a standard curve by taking the peak area value (A) as an ordinate (y) and the sample injection concentration (mu g/mL) as an abscissa (x). The standard curve of chlorogenic acid is y-26719 x +12380, R20.9991 (n-5), the linarin standard curve is:y＝19939x+15238，R20.9998(n is 5). Further result research shows that chlorogenic acid has good linear relation with corresponding peak area within the range of 30-155 mu g/mL, and linarin has good linear relation with corresponding peak area within the range of 12.3-62 mu g/mL.
(2) Specificity test
0.30011g of wild chrysanthemum granules are precisely added into 5mL of 50% methanol solution, stirred evenly and then kept stand for 10min, and filtered to obtain a sample solution. 0.30015g of sucrose powder is precisely added into 5mL of 50% methanol solution, stirred evenly and then kept stand for 10min, and filtered to be used as a blank control solution.
Sample solution, blank control solution, chlorogenic acid and linarin control solution were injected according to the method of example 3, and it can be seen from the results in fig. 1 that chromatographic peaks of chlorogenic acid and linarin in the sample result chart are clearly separated, and no miscellaneous peak interference is found in the blank control solution result chart.
(3) Sample application recovery test
3 parts of wild chrysanthemum granules are respectively taken, 5mL of prepared mixed reference substances (chlorogenic acid 42 mu g/mL and linarin 23.8 mu g/mL) are precisely added, the content is measured according to the method of the embodiment 3, and the recovery rate is calculated. The average recovery rate of chlorogenic acid is 99.70%, RSD is 0.44%, the average recovery rate of linarin is 96.67%, and RSD is 1.99%. As shown in tables 5-6.
TABLE 5 chlorogenic acid sample recovery results (n ═ 9)
TABLE 6 Mongolian glycosides sample recovery results (n ═ 9)
(4) Sample assay
Taking three parts of the wild chrysanthemum flower particles of the examples, the comparative examples 1 and the comparative examples 2 respectively, measuring according to the method of the example 3, recording peak areas, and calculating the content to obtain the table 7.
TABLE 7 results of content measurement of samples
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A method for measuring the content of chlorogenic acid and linarin is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of a reference solution: dissolving chlorogenic acid reference substance and linarin reference substance with solvent respectively, and metering volume to obtain the final product;
(2) sample solution preparation: grinding flos Chrysanthemi Indici granule, pretreating, adding methanol, heating under reflux, shaking, standing and filtering;
(3) determination of chromatographic conditions: the mobile phase is acetonitrile and phosphoric acid water solution with the mass fraction of 0.4 percent;
(4) and (3) determination: performing liquid chromatography on the sample solution and the reference solution to obtain the final product;
the pretreatment in the step (2) specifically comprises the steps of soaking the ground wild chrysanthemum flower particles in a mixed solution of ethanol and diethyl ether, drying, cooling, heating for the second time, and cooling to the normal temperature to obtain the wild chrysanthemum flower particles; the volume ratio of the ethanol to the diethyl ether is 4.5-5: 1; the liquid chromatography assay in step (4) specifically comprises gradient elution, and the elution method comprises:
0-10min, wherein the volume of the acetonitrile accounts for 13% -15% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 95% -85% of the total volume of the mobile phase;
10-15min, wherein the volume of the acetonitrile is increased from 13% -15% of the total volume to 30% -35%, and the volume of the acetonitrile is reduced from 95% -85% of the total volume to 80% -65%;
15-25min, wherein the volume of the acetonitrile accounts for 30-35% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 70-65% of the total volume of the mobile phase;
25-30min, the volume of the acetonitrile is reduced from 30% -35% of the total volume to 13% -15%, and the volume of the acetonitrile is increased from 70% -65% of the total volume to 87% -85%;
30-40min, wherein the volume of the acetonitrile accounts for 13-15% of the total volume of the mobile phase, and the volume of the phosphoric acid aqueous solution accounts for 87-85% of the total volume of the mobile phase;
the second heating is specifically to heat the mixture to 35-40 ℃ for the first time and to heat the mixture to 48-50 ℃ for the second time.
2. The method for measuring according to claim 1, wherein: the solvent in the step (1) is methanol solution.
3. The method for measuring according to claim 1, wherein: the concentration of the chlorogenic acid reference substance in the step (1) is 30-155 mu g/mL.
4. The method for measuring according to claim 1, wherein: the concentration of the linarin control in the step (1) is 12.3-62 mu g/mL.
5. The method for measuring according to claim 1, wherein: the concentration of the sample solution in the step (2) is 30-90 mg/mL.
6. The method for measuring according to claim 1, wherein: the theoretical plate number of chlorogenic acid in the chromatographic conditions of the liquid chromatography determination in the step (4) is not less than 12000, and the theoretical plate number of linarin is not less than 140000.
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