CN109307717B - Method for detecting content of polyphenol compounds in cannabis sativa oil - Google Patents
Method for detecting content of polyphenol compounds in cannabis sativa oil Download PDFInfo
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- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 34
- -1 polyphenol compounds Chemical class 0.000 title claims abstract description 12
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- 239000012071 phase Substances 0.000 claims abstract description 31
- 150000008442 polyphenolic compounds Chemical class 0.000 claims abstract description 31
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- 238000001514 detection method Methods 0.000 claims abstract description 25
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention relates to the technical field of chemical analysis, in particular to a method for detecting the content of polyphenol compounds in cannabis oil. The method comprises the following steps: mixing the cannabis oil and n-hexane, adding an extraction solvent, extracting by adopting an ultrasonic-assisted extraction technology, and purifying supernate to obtain a sample solution; respectively carrying out ultra-high performance liquid chromatography analysis on the sample solution and the standard solution, and obtaining the content of 9 polyphenols by adopting an external standard method; the conditions of the ultra-high performance liquid chromatography are as follows: the mobile phase A is methanol, the mobile phase B is 0.1 to 0.3 percent of acetic acid water solution, and gradient elution is carried out. The method adopts ultrasonic probe extraction, solid phase extraction purification and ultra-high performance liquid chromatography separation detection, realizes the rapid and accurate detection of 9 polyphenols in the cannabis oil, and can achieve good separation detection of 9 polyphenols only in 20 min.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for detecting the content of polyphenol compounds in cannabis oil.
Background
The cannabis sativa is an important medicinal and edible resource, and has a planting history of more than 6500 years in China. The hemp seed oil is also called as longevity oil, has obvious effects on reducing cholesterol, preventing cardiovascular and cerebrovascular diseases, resisting inflammation, aging, oxidation and atherosclerosis, and has good curative effect on curing mucosal skin wound after eye, nose and throat surgery. This is because the hemp oil is rich in polyunsaturated fatty acids, phytosterols, tocopherols and plant polyphenols. In recent years, a large number of research results show that the polyphenol component has good effects in various aspects such as oxidation resistance, antibiosis, antivirus, antimicrobial, blood fat regulation, blood sugar reduction and the like. The polyphenol in the vegetable oil can obviously improve the oxidation stability of the vegetable oil and improve the oil quality.
At home and abroad, there are few related researches on the determination of the content of polyphenol in the hemp oil. The existing method for measuring polyphenol in vegetable oil comprises a potassium permanganate titration method, a forinophenol method and a high performance liquid chromatography method. The potassium permanganate titration method and the folin phenol method are mainly used for measuring the content of total phenol, the variety and the content of polyphenol components cannot be determined, and the main method for measuring the polyphenol components is high performance liquid chromatography. Tea seed oil, rapeseed oil and olive oil are the most studied for polyphenol in vegetable oil in China. Because the types of the polyphenol compounds are more, the compositions are complex, the types and the contents of the polyphenol compounds in various vegetable oils are different, different pretreatment methods, mobile phase compositions and elution procedures can cause the difference of measurement results, and the method is suitable for measuring the polyphenol in one vegetable oil and is not suitable for the other method.
The existing method has the following problems: the polyphenol in the cannabis sativa oil is difficult to extract efficiently, the interference of a matrix is serious, the chromatographic separation time is long, the detection flux is low, and the like. Therefore, it is necessary to develop a method suitable for detecting the polyphenols in the cannabis oil.
Disclosure of Invention
In view of the above, the invention provides a method for detecting the content of polyphenol compounds in cannabis oil. The method can effectively and rapidly separate and accurately detect 9 polyphenols in the cannabis oil.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting the content of polyphenol compounds in cannabis oil, which comprises the following steps:
mixing the cannabis oil and n-hexane, adding an extraction solvent, extracting by adopting an ultrasonic-assisted extraction technology, and purifying supernate to obtain a sample solution;
preparing standard solution containing epicatechin, p-hydroxybenzoic acid, p-coumaric acid, naringin, 3, 4-dimethoxybenzoic acid, astragalin, daidzein, naringenin, and genistein;
respectively carrying out ultra-high performance liquid chromatography analysis on the sample solution and the standard solution, and obtaining the content of 9 polyphenols by adopting an external standard method;
the conditions of the ultra-high performance liquid chromatography are as follows: the mobile phase A is methanol, the mobile phase B is 0.1 to 0.3 percent of acetic acid water solution, and gradient elution is carried out;
the elution procedure was: 0-1.8 min, and the mobile phase B is 90-80%; 1.8-7 min, and the mobile phase B is 80-65%; 7-8.6 min, 65-50% of mobile phase B; 8.6-14.2 min, and the mobile phase B is 50-35%; 14.2-15.8 min, and the mobile phase B is 35-0 percent; 15.8-17.8 min, keeping the mobile phase B for 2.2min from 0-90%.
The method adopts ultrasonic probe extraction, solid phase extraction purification and ultra-high performance liquid chromatography separation detection, realizes the rapid and accurate detection of 9 polyphenols in the cannabis oil, and can achieve good separation detection of 9 polyphenols only in 20 min.
Preferably, mobile phase B is a 0.2% aqueous acetic acid solution.
Preferably, in the step of mixing the cannabis oil and the n-hexane, the mass-to-volume ratio of the cannabis oil to the n-hexane in g/mL is 1: 1.
Preferably, the mass-volume ratio of the cannabis oil to the extraction solvent is 1:2 in g/mL.
Preferably, the extraction solvent is 60 vt% -90 vt% methanol aqueous solution, 60 vt% -90 vt% ethanol aqueous solution or acetone.
Preferably, the extraction solvent is 60 vt% -90 vt% methanol aqueous solution or 60 vt% -90 vt% ethanol aqueous solution.
More preferably, the extraction solvent is 90 vt% aqueous methanol.
Preferably, the number of extractions is 2 to 4.
Preferably, the number of extractions is 3.
Preferably, the ultrasonic power of the ultrasonic-assisted extraction is 100-300W.
Preferably, the ultrasonic power of the ultrasonic-assisted extraction is 250W.
Preferably, the ultrasonic pulse mode is: the single-time ultrasonic treatment time is 3-9 s, and the intermittent time is 3-9 s.
In the specific embodiment provided by the present invention, the ultrasonic pulse mode is: single ultrasound time 6s, rest time 3 s.
In another embodiment provided by the present invention, the ultrasonic pulse mode is: single sonication time 9s, rest time 6 s.
In another embodiment provided by the present invention, the ultrasonic pulse mode is: single sonication time 3s, rest time 6 s.
In another embodiment provided by the present invention, the ultrasonic pulse mode is: single sonication time 9s, rest time 9 s.
Preferably, the temperature of the ultrasonic-assisted extraction is 20-30 ℃.
Preferably, the temperature of the ultrasound-assisted extraction is 25 ℃.
Preferably, the ultrasonic treatment time is 0.5-3 min.
Preferably, the ultrasonic treatment time is 1-3 min each time.
More preferably, each sonication time is 2 min.
Preferably, the purification is: respectively activating the solid phase extraction column with n-hexane and methanol, passing the extractive solution through the activated solid phase extraction column, collecting eluate, and concentrating by nitrogen blowing.
Preferably, the nitrogen is blown and concentrated to 1/14-1/6 of the total volume of the eluent.
Preferably, the solid phase extraction column is a diol-based extraction column, a C18 extraction column, a C8 solid phase extraction column, or a silica gel column.
Preferably, the solvent in the standard solution is methanol.
In the invention, the steps of preparing the standard solution of 9 phenolic compounds are as follows: respectively weighing 10mg of each standard substance in a 10mL brown volumetric flask, adding methanol to a constant volume, and uniformly mixing to obtain a standard substance with the concentration of 1 mg/mL; and respectively putting 200 mu L of each standard substance into a 25mL brown volumetric flask, adding methanol to a constant volume, and uniformly mixing to prepare a mixed standard solution. Then diluting step by step to prepare a series of mixed standard solutions.
Preferably, the standard solution and the sample solution are filtered through a 0.22 μm filter before they are subjected to liquid chromatography.
Preferably, the ultra high performance liquid chromatography system is equipped with a binary solvent manager, a sample manager, a column oven, a diode array detector.
Preferably, the parameters of the chromatographic column of the ultra-high performance liquid chromatography are as follows: 100 mm. times.2.1 mm, 1.7 μm.
Preferably, the ultra-high performance liquid chromatography adopts mobile phase equilibrium for 2min before each sample injection.
Preferably, the flow rate of the sample of the ultra-high performance liquid chromatography is 0.1-0.3 mL/min.
In the embodiment provided by the invention, the flow rate of the sample of the ultra-high performance liquid chromatography is 0.2mL/min,
preferably, the sample introduction amount of the ultra-high performance liquid chromatography is 4-6 muL.
In the embodiment provided by the invention, the sample introduction amount of the ultra-high performance liquid chromatography is 5 muL.
Preferably, the temperature of the column oven of the ultra-high performance liquid chromatography is 22-28 ℃.
In the embodiment provided by the invention, the temperature of the column incubator of the ultra-high performance liquid chromatography is 25 ℃.
Preferably, the detection wavelength of the ultra-high performance liquid chromatography is 275-285 nm.
Preferably, the detection wavelength is 280 nm.
Preferably, in the standard solution, the mass volume concentration of epicatechin is 0.42-13.44 mug/mL, the mass volume concentration of p-hydroxybenzoic acid is 0.23-7.38 mug/mL, the mass volume concentration of p-coumaric acid is 0.19-6.07 mug/mL, the mass volume concentration of naringin is 0.19-6.00 mug/mL, the mass volume concentration of 3, 4-dimethoxybenzoic acid is 0.20-6.24 mug/mL, the mass volume concentration of astragalin is 0.30-9.70 mug/mL, the mass volume concentration of daidzein is 0.19-6.18 mug/mL, the mass volume concentration of naringenin is 0.34-10.80 mug/mL, and the mass volume concentration of genistein is 0.30-9.70 mug/mL.
In the present invention, the procedure for preparing the standard curve of 9 phenolic compounds is as follows: and (3) analyzing the series of mixed standard substance solutions by using an ultra high performance liquid chromatograph, taking the mass concentration of each phenolic compound as a horizontal coordinate (X) and correspondingly taking the chromatographic peak area as a vertical coordinate (Y), performing linear fitting, and drawing a standard curve. The standard curves of the 9 phenolic compounds were obtained.
In quantification, the following formula is used for calculation:
in the formula:
x-the amount of phenolic compounds in the sample in micrograms per hundred grams;
v is the volume of the phenolic compound sample after being extracted and liquid nitrogen is blown and concentrated, and is 2 mL;
c-the concentration of polyphenols in the sample (found by standard curve) in micrograms per milliliter;
m is the mass of the sample in grams.
The invention provides a method for detecting the content of polyphenol compounds in cannabis oil. The method comprises the following steps: mixing the cannabis oil and n-hexane, adding an extraction solvent, extracting by adopting an ultrasonic-assisted extraction technology, and purifying supernate to obtain a sample solution; respectively carrying out ultra-high performance liquid chromatography analysis on the sample solution and the standard solution, and obtaining the content of 9 polyphenols by adopting an external standard method; the conditions of the ultra-high performance liquid chromatography are as follows: the mobile phase A is methanol, the mobile phase B is 0.1 to 0.3 percent of acetic acid water solution, and gradient elution is carried out. The invention has at least one of the following advantages:
1. the method adopts ultrasonic probe extraction, solid phase extraction purification and ultra-high performance liquid chromatography separation detection, realizes the rapid and accurate detection of 9 polyphenols in the cannabis oil, and can achieve good separation detection of 9 polyphenols only in 20 min.
2. In the detection method, the linear relation of various phenolic compounds is good in a corresponding linear range, and the correlation coefficient reaches more than 0.99; the RSD of the invention is between 0.43 and 2.03, and the recovery rate is 92 to 104 percent. High precision, accurate result and less background interference.
Drawings
Fig. 1 shows UPLC diagrams of standard solutions of 9 polyphenols, wherein 1: p-hydroxybenzoic acid; 2: epicatechin; 3: p-coumaric acid; 4: naringin; 5: 3. 4-dimethoxybenzoic acid; 6: astragalin; 7: daidzein; 8: naringenin; 9: genistein;
FIG. 2 shows a UPLC profile of polyphenols in cannabis oil, wherein 1: p-hydroxybenzoic acid; 2: epicatechin; 3: p-coumaric acid; 4: naringin; 5: 3. 4-dimethoxybenzoic acid; 6: astragalin; 7: daidzein; 8: naringenin; 9: genistein.
Detailed Description
The invention discloses a method for detecting the content of polyphenol compounds in cannabis oil, which can be realized by appropriately improving process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The reagent or instrument used in the method for detecting the content of the polyphenol compounds in the cannabis oil can be purchased from the market.
Reagent: the 9 phenolic compound standards comprise epicatechin, p-hydroxybenzoic acid, p-coumaric acid, naringin, 3, 4-dimethoxybenzoic acid, kaempferol-3-O-glucoside, daidzein, naringenin and genistein, and the purity is more than or equal to 98 percent; acetic acid and methanol are chromatographically pure, and normal hexane and DMF are analytically pure.
The instrument comprises the following steps: the device comprises an ultrasonic instrument, a vortex instrument, a centrifugal machine, a solid phase extraction device, a nitrogen blowing device and an ultra-high performance liquid chromatography system.
The invention is further illustrated by the following examples:
example 1
a. The preparation method of the mixed standard solution of the 9 phenolic compounds comprises the following steps: respectively weighing 10mg of each standard substance (epicatechin, p-hydroxybenzoic acid, p-coumaric acid, naringin, 3, 4-dimethoxybenzoic acid, kaempferol-3-O-glucoside, daidzein, naringenin and genistein) in a 10mL brown volumetric flask, adding methanol to a constant volume, and uniformly mixing to obtain the standard substance with the concentration of 1 mg/mL. And respectively putting 200 mu L of each standard substance into a 25mL brown volumetric flask, adding methanol to a constant volume, and uniformly mixing to prepare a mixed standard solution. And then diluting step by step to prepare a series of mixed standard solutions, and performing HPLC analysis to draw a regression curve of the standard substance.
The chromatographic conditions are as follows:
a chromatographic column: c18 ultra high performance liquid chromatography column;
mobile phase A: methanol;
mobile phase B: 0.2% aqueous acetic acid;
flow rate: 0.2 mL/min;
sample introduction amount: 5 mu L of the solution;
wavelength: 280 nm;
column temperature: 25 ℃;
gradient elution: 0-1.8 min, 90-80% of mobile phase B, 1.8-7 min and 80-65% of mobile phase B; 7-8.6 min, 65-50% of mobile phase B; 8.6-14.2 min, and the mobile phase B is 50-35%; 14.2-15.8 min, and the mobile phase B is 35-0 percent; 15.8-17.8 min, keeping the mobile phase B for 2.2min from 0-90%. The total time is 20 min. Equilibrate for 2min before each injection.
The ultra-high performance liquid chromatogram of the mixed standard solution of 9 phenolic compounds is shown in figure 1.
b. The 9 polyphenols were added to the cannabis oil at 1, 2 times the level of phenolic compounds in the cannabis oil, 3 times for each level of spiking, the content of the 9 polyphenols in the spiked samples was determined and the recovery and relative standard deviation calculated as shown in table 1.
c. 2g of cannabis oil 1 is accurately weighed, 2mL of n-hexane is added to fully dissolve the cannabis oil, and 4mL of 90% methanol is added. Performing ultrasonic extraction for 2min under the conditions of ultrasonic power of 250W, ultrasonic pulse mode of 6s/3s and temperature of 25 ℃, taking supernatant, adding 4mL of 90% methanol, performing repeated extraction under the same ultrasonic conditions, taking supernatant, and repeating for 2 times. The supernatants were combined.
d. Respectively activating a diol-based solid phase extraction column by using 4mL of n-hexane and methanol, passing the polyphenol extracting solution through the solid phase extraction column, collecting eluent, and blowing and concentrating the eluent to 2mL by using nitrogen.
e. And carrying out ultra performance liquid chromatography analysis on the sample, wherein the chromatographic conditions are the same as those of the standard solution.
f. The chromatogram of cannabis oil is shown in FIG. 2. And comparing the chromatogram of the sample solution with the standard solution curve to obtain the content of various phenolic substances in the cannabis oil.
The linear regression equation, correlation coefficient, detection limit, relative standard deviation and recovery for the 9 phenolic compounds are shown in table 1.
Table 1: the results of the standard curve linear regression equation, correlation coefficient, detection limit, relative standard deviation and recovery rate in example 1 of the present invention.
In a corresponding linear range, the linear relation of various phenolic compounds is good, and the correlation coefficient reaches more than 0.99; the RSD of the invention is between 0.43 and 2.03, and the recovery rate is 92 to 104 percent. High precision and accurate result.
Example 2:
a. 3g of cannabis oil 2 is accurately weighed, 3mL of n-hexane is added to fully dissolve the cannabis oil, and 6mL of 60% methanol is added. Performing ultrasonic extraction for 1min under the conditions of ultrasonic power of 300W, ultrasonic pulse mode of 9s/6s and temperature of 30 ℃, taking supernatant, adding 6mL of 60% methanol, performing repeated extraction under the same ultrasonic conditions, taking supernatant, and repeating for 2 times. The supernatants were combined.
b. Respectively activating C18 solid phase extraction column with 5mL n-hexane and methanol, passing the polyphenol extractive solution through the solid phase extraction column, collecting eluate, and concentrating to 2mL by nitrogen blowing.
c. The sample was subjected to ultra performance liquid chromatography under the same chromatographic conditions as in example 1.
d. And comparing the chromatogram of the sample solution with the standard solution curve to obtain the content of various phenolic substances in the cannabis oil.
Example 3:
a. accurately weighing 4g of cannabis sativa oil 3, adding 4mL of n-hexane for full dissolution, and adding 8mL of 90% ethanol. Performing ultrasonic extraction for 2min under the conditions of ultrasonic power of 100W, ultrasonic pulse mode of 3s/6s and temperature of 30 ℃, taking supernatant, adding 8mL of 90% ethanol, performing repeated extraction under the same ultrasonic conditions, taking supernatant, and repeating for 3 times. The supernatants were combined.
b. Respectively activating C8 solid phase extraction column with 3mL n-hexane and methanol, passing the polyphenol extractive solution through the solid phase extraction column, collecting eluate, and concentrating to 2mL by nitrogen blowing.
c. The sample was subjected to ultra performance liquid chromatography under the same chromatographic conditions as in example 1.
d. And comparing the chromatogram of the sample solution with the standard solution curve to obtain the content of various phenolic substances in the cannabis oil.
Example 4:
a. 2.5g of hemp oil 4 is accurately weighed, 2.5mL of n-hexane is added to be fully dissolved, and 5mL of 60% ethanol is added. Performing ultrasonic extraction for 3min under the conditions of ultrasonic power of 200W, ultrasonic pulse mode of 9s/9s and temperature of 20 ℃, taking supernatant, adding 5mL of 60% ethanol, performing repeated extraction under the same ultrasonic conditions, taking supernatant, and repeating for 1 time. The supernatants were combined.
b. Activating silica gel solid phase extraction column with 6mL of n-hexane and methanol respectively, passing polyphenol extract through the solid phase extraction column, collecting eluate, and concentrating to 2mL by nitrogen blowing.
c. The sample was subjected to ultra performance liquid chromatography under the same chromatographic conditions as in example 1.
d. And comparing the chromatogram of the sample solution with the standard solution curve to obtain the content of various phenolic substances in the cannabis oil.
The raw materials listed in the invention, the upper and lower limits and interval values of the raw materials of the invention, and the upper and lower limits and interval values of the process parameters (such as temperature, time and the like) can all realize the invention, and the examples are not listed.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for detecting the content of polyphenol compounds in cannabis oil is characterized by comprising the following steps:
mixing the cannabis oil and n-hexane, adding an extraction solvent, extracting by adopting an ultrasonic-assisted extraction technology, and purifying supernate to obtain a sample solution;
preparing standard solution containing epicatechin, p-hydroxybenzoic acid, p-coumaric acid, naringin, 3, 4-dimethoxybenzoic acid, astragalin, daidzein, naringenin, and genistein;
respectively carrying out ultra-high performance liquid chromatography analysis on the sample solution and the standard solution, and obtaining the content of 9 polyphenols by adopting an external standard method;
the conditions of the ultra-high performance liquid chromatography are as follows: c18 ultra high performance liquid chromatography column, gradient elution, wherein the mobile phase A is methanol, the mobile phase B is 0.1% -0.3% acetic acid water solution;
the elution procedure was: 0-1.8 min, and the mobile phase B is 90-80%; 1.8-7 min, and the mobile phase B is 80-65%; 7-8.6 min, 65-50% of mobile phase B; 8.6-14.2 min, and the mobile phase B is 50-35%; 14.2-15.8 min, and the mobile phase B is 35-0 percent; 15.8-17.8 min, keeping the mobile phase B for 2.2min from 0-90%.
2. The detection method according to claim 1, wherein the extraction solvent is 60 vt% to 90 vt% methanol aqueous solution, 60 vt% to 90 vt% ethanol aqueous solution, or acetone, and the number of times of extraction is 2 to 4.
3. The detection method according to claim 1, wherein the ultrasonic power of the ultrasonic-assisted extraction is 100-300W, and the ultrasonic pulse mode is as follows: the single ultrasonic time is 3-9 s, and the intermittent time is 3-9 s; the temperature of ultrasonic-assisted extraction is 20-30 ℃, and the ultrasonic time is 0.5-3 min each time.
4. The detection method according to claim 1, wherein the purification is: respectively activating the solid phase extraction column with n-hexane and methanol, passing the extractive solution through the activated solid phase extraction column, collecting eluate, and concentrating by nitrogen blowing.
5. The detection method according to claim 4, wherein the solid phase extraction column is a diol-based extraction column, a C18 extraction column, a C8 solid phase extraction column, or a silica gel column.
6. The detection method according to claim 1, wherein the solvent in the standard solution is methanol.
7. The detection method according to claim 1, wherein the parameters of the chromatographic column of the ultra-high performance liquid chromatography are: 100 mm. times.2.1 mm, 1.7 μm.
8. The detection method according to claim 1, wherein the flow rate of the sample for the ultra-high performance liquid chromatography is 0.1 to 0.3mL/min, the amount of the sample is 4 to 6 μ L, and the temperature of the column oven is 22 to 28 ℃.
9. The detection method according to claim 1, wherein the detection wavelength of the ultra-high performance liquid chromatography is 275 to 285 nm.
10. The detection method according to claim 1, wherein in the standard solution, the mass volume concentration of epicatechin is 0.42 to 13.44 μ g/mL, the mass volume concentration of p-hydroxybenzoic acid is 0.23 to 7.38 μ g/mL, the mass volume concentration of p-coumaric acid is 0.19 to 6.07 μ g/mL, the mass volume concentration of naringin is 0.19 to 6.00 μ g/mL, the mass volume concentration of 3, 4-dimethoxybenzoic acid is 0.20 to 6.24 μ g/mL, the mass volume concentration of astragalin is 0.30 to 9.70 μ g/mL, the mass volume concentration of daidzein is 0.19 to 6.18 μ g/mL, the mass volume concentration of naringenin is 0.34 to 10.80 μ g/mL, and the mass volume concentration of genistein is 0.30 to 9.70 μ g/mL.
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