CN112362777A - Microanalysis method of symbolic component Borojo-8, 9-iridoid glycoside in Borojo and products thereof - Google Patents
Microanalysis method of symbolic component Borojo-8, 9-iridoid glycoside in Borojo and products thereof Download PDFInfo
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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
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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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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- 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
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- G01N30/72—Mass spectrometers
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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Abstract
The invention discloses a microanalysis method of a symbolic component Borojo-8, 9-iridoid glycoside in Borojo and products thereof, which comprises the following steps: step 1: pretreating a Borojo raw fruit; step 2: pretreating Borojo enzymolysis concentrated powder; and step 3: pretreating the boletus fermented fruit wine; and 4, step 4: pretreating the beverage containing the Bordeaux mixture; and 5: pretreatment of cakes, bread and biscuits containing the Bordeaux mixture; step 6: pretreatment of cosmetic containing Bordeaux mixture. The invention analyzes and tests the trace marker active substances in the tylophora fruits with different matrixes, eliminates the interference of similar components in different products, and introduces the use of internal standards in mass spectrometry. Meanwhile, a liquid chromatography analysis technology is established, and the reappearance analysis of the trace target substances in the Baoleguo product and the accuracy of the result are ensured.
Description
Technical Field
The invention relates to a determination method, in particular to a microanalysis method of a marking component Borojo-8, 9-iridoid glycoside in Borojo and products thereof.
Background
Bordeaux (Chinese name) (Spanish name Borojo) is a fruit grown in the tropical rainforest of south America, which is only suitable for growing in the Ecuador climate of south America, plants of the genus Rubiaceae, Linum. The diameter of the Borojo fruit reaches 7-12 cm, the Borojo fruit is green when immature, the Borojo fruit turns into dark brown after mature, the weight is 1000 g/fruit, the fruit pulp is soft, sour and viscous, and the weight of the fruit pulp accounts for 88% after seeds are removed. The Borojo has a special unique fragrance, and is strong and long-lasting. For a long time, local native people regard the native people as treasure due to the effects of rich nutrition and health promotion, and have eating habits for thousands of years. The Baoleguo is rich in nutrition, contains components such as polysaccharide, polyphenol, total flavone, terpenoids, vegetable oil, various mineral substances, various vitamins, rare earth elements, protein, essential amino acid and the like, has a similar preparation ratio of nutrient components to human beings, is suitable for being absorbed and utilized by human beings, and is called as '21 st century super fruit' by Americans.
Through international search, 2008 reported by the food technology development center at the new jersey state university (RUTGERS), a substance with molecular weight of 388 was found in the Borojo plant map. Considered to be the only previously unidentified, abundant, novel, "single polyphenolic" substance. The laboratory optimization adopts ISO14502-1/2:2005 method for testing tea polyphenols in SOP plants- -spectrophotometry, 0.323g of tea polyphenols (gallic acid in gallic acid) is detected from 100g of Borojo sorbent Cuatrec freeze-dried powder.
The website www.nutropical.com on 8/11/2010 reports an article by Secaucus NJ (PRWEB): the research result of Borojo sorbent Cuatrec shows that the Borojo sorbent Cuatrec only contains one polyphenol and has high relative concentration, which is unique and revolutionary significance in the plant world.
In 2010, a food research technical team taking the red-brown teaching as the core is entrusted by south American Chinese and has comprehensively researched the total nutrient components, food safety and nutritional efficacy of Borojo sorbilis Cuatrec. In 2013, the plant is cooperated with a south China botanical garden of Chinese academy of sciences, and Borojo sorbilis Cuatrec is identified and named as the Chinese named Baoleguo. Application of the Baoleguo powder in 2014 enters the administrative approval of new food raw materials in China. The No. 7 bulletin was approved by the national health council in 2017 as: chinese new food raw material Baoleguo powder.
As early as 2011, a pyrogen team optimally extracts substances which have extremely high abundance and single molecular weight and are turned into polyphenol by American scholars from the tylosin, and the molecular structure of the high-content natural active substances in the tylosin is identified by adopting 3 methods of high-resolution mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. The results were obtained:
1. high resolution mass spectrometry HPLC-MS/MS full scan, instrument: API 3200Qtrap, ion scan mode: EI (El)
ESI-anion scan, retention time 2.79min, reproducible (RUTGERS) food technology development center confirmed as "single polyphenols" components, molecular weight 388. In the scanning spectrum, the polarity of the substance is the strongest. There was also one M/z 775.5 species in the co-efflux material, which was inferred to be 388 dimer 775.5(2M-H), as shown in FIG. 1.
② the special scan of the dimer M/Z775.5 confirms that the secondary fragment peak is judged to be M/Z387 (M-H-), and the decarboxylation peak is judged to be M/Z343 (M-C02-H-).
And thirdly, carrying out special scanning confirmation on M/Z387 (M-H-) and M/Z343 (M-H20-H-) and all fragments thereof, and deducing that the six-carbon sugar can be removed from the M/Z343 (M-H20-H-). The abundance of this scanning effect is far superior to the fragment peak pattern of the (RUTGERS) food technology development center.
Analysis of query mass spectrometry literature shows that after the molecular weight of hexose 162 is removed from structures such as polyphenol, flavone or flavonoid, the generated skeleton structure is larger than 181, but no such substance exists in the ion trap scanning result, so that preliminary judgment shows that the active substance with the molecular weight of 388, which is the main characteristic substance in the tylophora fruit, is not a polyphenol, flavone or flavonoid structure.
The iridoid glycoside has the characteristic of terminal six-carbon sugar and has a strong [ M-H-162 ] -peak in a CID spectrum. In our scan, M/Z181 is subjected to peak M/Z343 (from M-C02-H-decarboxylation, M/Z181 can be subjected to decarboxylation to form M/Z136.9 and 93. the tylophora has the remarkable characteristic of iridoid glycoside mass spectrum.
Sixthly, according to the characteristics that the pH value of the tylophora fruit is slightly acidic and the retention time of the M/Z387 is short, the target substance is supposed to have a plurality of carboxylic acid structures except the hexose, at least M/Z181 can be continuously decarboxylated to form M/Z136.9 and 93, and two carboxylic acid groups may be contained.
And finally, confirming that the accurate molecular weight of the original 388-molecular weight substance is M/Z387.09289 and the molecular formula is C16H 20O 11 under the high-resolution FTMS mass spectrometry. Another decarboxylated characteristic peak M/Z343 has an exact molecular weight of M/Z309.04361 and a molecular formula of C15H 19O 10.
2. Molecular structure identification result of active substance with characteristic molecular weight (388) by nuclear magnetic resonance
Nuclear magnetic resonance spectrometer, model: avance 500.
Identification of structures of ChemDraw Ultra inferred Hydrogen and carbon spectra
As shown in fig. 2, from the nuclear magnetic resonance hydrogen spectrum, the structure of the main active substance of the tylophora theoretically predicted to be basically coincident with the actual hydrogen spectrum in the full scan. The H spectrum also has a peak that should be around 5.1, which is not present at this time, and may be masked by the highest solvent peak, 5.0. The actual hydrogen spectrum can determine that other structures such as aromatic rings do not exist although the interference signal of the deuterated methanol exists. Therefore, it can be confirmed that there is no aromatic ring structural substance in the hydrogen spectrum.
The carbon spectrum of the nuclear magnetic resonance spectrometer basically conforms to the structure predicted by the full-scan mass spectrometry theory. In the actual carbon spectrum of the primary active of tylophora, each bar represents one carbon atom and a standard six carbon sugar cluster appears. Namely (C '1-98.994, C' 2-73.219, C '3-76.379, C' 4-70.015, C '5-76.730 and C' 6-61.260). The distribution of carbon atoms on the main backbone of glycosylated 8, 9-iridoid glycosides (8,9-unsaturated iridoids) is: c1-95.022, C3-152.280, C4-111.141, C5-38.849, C6-146.416, C7-134.825, C8-33.89, C9-45.977, C10-166.742 and C11-169.242. The others (47.166-48.189) are 2N +1 peaks of deuterated methanol, which are interference signals of the reagent.
3. And (3) infrared spectrum identification: fourier infrared-raman-microscopy (FT-IP) results:
as shown in FIG. 3, infrared spectrum detection is performed by using the extracted pure molecular weight crystal of Bordeaux, and it can be concluded that 3405.21 is a free-OH group, 2890 is a-CH-group, 1250-.
The structure of the active substance is analyzed and deduced through mass spectrum, nuclear magnetic resonance and infrared spectrum, and the following can be determined: the molecular structure of the main active substance (mass spectrum mass molecule number 388 substance) in the tylophora floribunda is as follows:
chemical name: 8, 9-glycosylated-iridoid glycosides (8, 9-unsatus iridoids) (sesquiterpenes)
The molecular formula is as follows: C16H 20O 11
Precise molecular weight: M/Z387.09289
Chemical structural formula:
it was therefore concluded that: the main active substance of Bordeaux is sesquiterpene rather than polyphenol. This is different from the results reported in the laboratories of the food engineering center of america.
The technology is granted by patent invention ZL201110236556.7 from patent invention 2013.12.18 of patent invention of Borojo active extract, preparation method and antibacterial and antioxidant application in 2011.
In the technology, the active substance, namely the bolojo-iridoid glycoside, in the Borojo jam, the fruit powder and the primary products thereof is quantitatively tested by an external standard method by adopting an HPLC-MS/MS method, and a quantitative analysis method for the active substance in the bolojo jam and the primary products thereof is established. And is used as a method for identifying the authenticity of the Borojo and the marking property of the product thereof.
In the process of continuously applying and researching the tylophora fruits and developing products, the technology finds that the extraction of trace tylophora fruits active ingredients in health-care food, cosmetics, wine, beverage and food samples with complex matrixes is easily interfered by other ingredients in the samples in the analysis process. And the cost of using the mass spectrum external standard method is high.
Disclosure of Invention
The invention aims to provide a microanalysis method of a marked component Borojo-8, 9-iridoid glycoside in Borojo and products thereof, tests trace active matters in various different matrix Borojo products, eliminates interference of similar components in the products, and introduces the use of an internal standard in a mass spectrometry. Meanwhile, a liquid chromatography analysis technology is established, so that the analysis reappearance of the target substances of most conventional samples and the accuracy of the result are ensured under the prospect of continuously expanding the application range of the tylosin, and the problems in the background technology can be solved.
In order to achieve the purpose, the invention provides the following technical scheme:
the microanalysis method of the marking component Borojo-8, 9-iridoid glycoside in the Borojo and the products thereof comprises the following steps:
step 1: pretreatment of Borojo raw fruit
Weighing 10g of sample into a beaker, adding 100mL of 60% methanol water, stirring uniformly, sealing the mouth of the beaker by using tinfoil paper, heating in a water bath at 90 ℃ for 30 minutes, taking out the beaker, cooling to room temperature, centrifuging to obtain supernatant, filtering by using an ultrafiltration membrane of 0.22 mu m, and detecting by using a high performance liquid chromatograph;
step 2: pretreatment of Borojo enzymolysis concentrated powder
Weighing 1.0g of sample into a beaker, dissolving with 60% methanol to a constant volume of 100ml, shaking up, filtering with an ultrafiltration membrane of 0.22 μm, diluting the filtrate 1000 times with 60% methanol water solution, and detecting with a high performance liquid chromatograph;
and step 3: pretreatment of Bordeaux fermented fruit wine
Weighing 1.0g of sample in a beaker, carrying out water bath in hot water at 60 ℃ for 20min, volatilizing ethanol in the sample, reducing the temperature of the sample liquid to room temperature, metering the volume to 25ml by using 60% methanol water solution, shaking up, filtering by using an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times by using 60% methanol water solution, and detecting by using a high performance liquid chromatograph;
and 4, step 4: pretreating the beverage containing the Bordeaux mixture;
and 5: pretreatment of cake, bread and biscuit containing Bordeaux mixture
Crushing or shearing a sample by using a crusher, fully and uniformly stirring, weighing about 1.0g of sample in a beaker, adding 20ml of petroleum ether, uniformly stirring, standing for layering, pouring off a petroleum ether layer, repeating the operation for 2 times, fully removing fat in the sample, volatilizing the petroleum ether, adding about 20ml of 60% methanol aqueous solution, ultrasonically extracting for 30min, transferring the upper layer solution into a 25ml volumetric flask after the solution is cooled to room temperature, washing residues by using a small amount of 60% methanol aqueous solution, merging a washing solution into the volumetric flask, fixing the volume to scale by using the 60% methanol aqueous solution, uniformly shaking, and detecting by using a high performance liquid chromatograph;
step 6: pretreatment of cosmetic containing Bordeaux mixture.
Further, step 4 comprises two parts of the processing of the liquid sample and the processing of the solid sample, wherein:
(1) pretreatment of liquid samples
Weighing 1.0g of sample in a 25ml volumetric flask, adding about 20ml of 60% methanol aqueous solution, carrying out ultrasonic extraction for 10min, cooling the liquid to room temperature, fixing the volume to the scale with the 60% methanol aqueous solution, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times with the 60% methanol aqueous solution, and detecting with a high performance liquid chromatograph;
(3) pretreatment of solid samples
Weighing 0.5g-1.0g of sample in a 25ml volumetric flask, adding about 5.0ml of water, performing vortex oscillation, fully dissolving the sample, adding 15.0ml of 60% methanol aqueous solution after dissolution, shaking up, performing ultrasonic extraction for 10min-15min, cooling the liquid to room temperature, fixing the volume to a scale by using the 60% methanol aqueous solution, shaking up, filtering by using an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times by using the 60% methanol aqueous solution, and detecting by using a high performance liquid chromatograph.
Further, step 6 comprises two parts of the treatment of the non-waxy sample and the treatment of the waxy sample, wherein:
(1) treatment of wax-free samples
Weighing 2.5g of sample in a 10.0ml colorimetric tube, dissolving with 5.0ml of purified water, performing ultrasonic treatment for 30 minutes, cooling the temperature of a test solution to room temperature, metering the volume to a scale with 60% methanol water, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
(2) pretreatment of waxy samples
Weighing 2.5g of sample in a 10.0ml colorimetric tube, dissolving with 1ml of tetrahydrofuran, adding 4ml of methanol, vibrating on a vortex oscillator at high speed for 5min, carrying out ultrasonic treatment for 30min, cooling the temperature of the test solution to room temperature, metering the volume to scale with the methanol, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph.
Further, the method comprises the following steps:
s1: pretreatment of Borojo raw fruit
Weighing 1.0g of sample into a beaker, adding 2.0mL of internal standard solution, adding 100mL of 60% methanol water, stirring uniformly, sealing the opening of the beaker by using tinfoil paper, heating in a water bath at 90 ℃ for 30 minutes, taking out the beaker, cooling to room temperature, centrifuging to obtain supernatant, filtering by using an ultrafiltration membrane of 0.22 mu m, and detecting by using a high performance liquid chromatography-mass spectrometer;
s2: pretreatment of Borojo enzymolysis concentrated powder
Weighing 0.1g of sample into a beaker, adding 2.0ml of internal standard use solution, dissolving with 60% methanol to constant volume to 100ml, shaking up, filtering with 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer;
s3: pretreatment of Bordeaux fermented fruit wine
Weighing 1.0g of sample in a beaker, adding 0.5ml of internal standard solution, carrying out water bath in hot water at 60 ℃ for 20min, volatilizing ethanol, cooling the temperature of the sample solution to room temperature, then using 60% methanol water to fix the volume to 25ml, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
s4: pretreatment of beverage containing Bordeaux mixture
S5: pretreatment of cake, bread and biscuit containing Bordeaux mixture
Crushing or shearing a sample by using a crusher, fully and uniformly stirring, weighing about 1.0g of sample in a beaker, adding 20ml of petroleum ether, uniformly stirring, standing for layering, pouring off a petroleum ether layer, repeating the operation for 2 times, fully removing fat in the sample, volatilizing the petroleum ether, adding about 20ml of 60% methanol aqueous solution, adding 0.5ml of internal standard solution, ultrasonically extracting for 30min, cooling the solution to room temperature, transferring the upper solution to a 25ml volumetric flask, washing residues by using a small amount of 60% methanol aqueous solution, merging a washing solution into the volumetric flask, fixing the volume to the scale by using the 60% methanol aqueous solution, shaking uniformly, and detecting by using a high performance liquid chromatograph;
s6: pretreatment of cosmetic containing Bordeaux mixture.
Further, S4 includes two parts of the processing of the liquid sample and the processing of the solid sample, wherein:
a: pretreatment of liquid samples
Weighing 1.0g of sample in a 25ml volumetric flask, adding 0.5ml of internal standard use solution, adding about 20ml of 60% methanol aqueous solution, carrying out ultrasonic extraction for 10min, cooling the liquid to room temperature, fixing the volume to the scale with the 60% methanol aqueous solution, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
b: pretreatment of solid samples
Weighing 0.5g-1.0g of sample in a 25ml volumetric flask, adding about 5.0ml of water, performing vortex oscillation, fully dissolving the sample, adding 0.5ml of internal standard use solution, adding 15.0ml of 60% methanol aqueous solution after dissolution, shaking up, performing ultrasonic extraction for 10min-15min, cooling the liquid to room temperature, metering to the scale with 60% methanol aqueous solution, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatograph.
Further, S6 includes treatment of a non-waxy sample and treatment of a waxy sample, wherein:
a: pretreatment of wax-free samples
Weighing 2.5g of sample in a 10ml colorimetric tube, adding 0.2ml of internal standard use solution, dissolving with 4.0ml of purified water, carrying out ultrasonic treatment for 30 minutes, cooling the temperature of the test solution to room temperature, fixing the volume to the scale with 60% methanol water, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer;
b: pretreatment of waxy samples
Weighing 2.5g of sample into a 10ml colorimetric tube, adding 0.2ml of internal standard use solution, dissolving with 1ml of tetrahydrofuran, adding 4.0ml of methanol, vibrating on a vortex oscillator at high speed for 5min, carrying out ultrasonic treatment for 30min, cooling the temperature of the test solution to room temperature, fixing the volume to the scale with methanol, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer.
Compared with the prior art, the invention has the beneficial effects that:
1. the 2 detection methods provided by the invention have higher detection sensitivity and specificity than the first method.
2. The method introduces apigenin as an internal standard substance of a mass spectrum internal standard method, can offset system errors caused by the change of a sample in the processing process, the sample loading volume, even a mobile phase, a detector and the like, and ensures the accuracy of a detection result.
3. The 2 detection methods provided by the invention have the advantages of low cost, safety, environmental protection and extremely high economic value.
Drawings
FIG. 1 is a scanning spectrum of a high resolution mass spectrometry HPLC-MS/MS full scan of the prior art;
FIG. 2 is a prior art NMR hydrogen spectrum;
FIG. 3 is a prior art infrared spectrum;
FIG. 4 is a high performance liquid chromatogram of Borojo-iridoid glycoside according to the present invention;
FIG. 5 is a drawing of a reference sample of Borojo-iridoid glycoside and an internal standard substance according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Microanalysis method (high performance liquid chromatography) for Borojo-8, 9-iridoid glycoside as marker component in Borojo and its product
1. Reagents and solutions
The experimental water is first-grade water meeting the GB/T6682 specification.
Reagent: acetonitrile (chromatographically pure); methanol (chromatographically pure); tetrahydrofuran (chromatographically pure); petroleum ether (analytically pure, boiling point 30-60 ℃), standard: borojo control.
Solution preparation:
1. standard solution
1.1 Standard stock solutions
The Borojo control, 10mg, was weighed accurately and placed in a 10mL brown volumetric flask and made up with 60% methanol in water to a stock solution concentration of 1.0 mg/mL. The stock solutions were protected from light by tinfoil and stored in a refrigerator at 4 ℃.
1.2. Standard use solution
According to the following steps: 10. 1: 100. 1:200, 1: 1,000, 1:2,000, 1: the stock solution of standard was diluted with 50% methanol water at a ratio of 10,000 into a 2mL sample vial, standard curve concentration range: 0.1 mu g/ml to 100 mu g/ml.
2. Apparatus and device
2.1 HPLC model number of Shimadzu corporation: LC20A
2.2 common C18 reversed phase column, specification: 150 x 4.6 mm; particle size: 5 mu
2.3 constant temperature water bath
2.4 electronic balance
2.5 ultrasonic cleaning instrument
2.6 vortex oscillator.
The experimental procedure for this example is as follows:
step 1: pretreatment of Borojo raw fruit
Weighing 10g of sample into a beaker, adding 100mL of 60% methanol water, stirring uniformly, sealing the mouth of the beaker by using tinfoil paper, heating in a water bath at 90 ℃ for 30 minutes, taking out the beaker, cooling to room temperature, centrifuging to obtain supernatant, filtering by using an ultrafiltration membrane of 0.22 mu m (or filtering by using filter paper, and filtering the filtrate by using the ultrafiltration membrane), and detecting by using a high performance liquid chromatograph;
step 2: pretreatment of Borojo enzymolysis concentrated powder
Weighing 1.0g of sample into a beaker, dissolving with 60% methanol to a constant volume of 100ml, shaking up, filtering with an ultrafiltration membrane of 0.22 μm, diluting the filtrate 1000 times with 60% methanol water solution, and detecting with a high performance liquid chromatograph;
and step 3: pretreatment of Bordeaux fermented fruit wine
Weighing 1.0g of sample in a beaker, carrying out water bath in hot water at 60 ℃ for 20min, volatilizing ethanol in the sample, reducing the temperature of the sample liquid to room temperature, metering the volume to 25ml by using 60% methanol water solution, shaking up, filtering by using an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times by using 60% methanol water solution, and detecting by using a high performance liquid chromatograph;
and 4, step 4: pretreatment of beverage containing Bordeaux mixture
(1) Pretreatment of liquid samples
Weighing 1.0g of sample in a 25ml volumetric flask, adding about 20ml of 60% methanol aqueous solution, carrying out ultrasonic extraction for 10min, cooling the liquid to room temperature, fixing the volume to the scale with the 60% methanol aqueous solution, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times with the 60% methanol aqueous solution, and detecting with a high performance liquid chromatograph;
(2) pretreatment of solid samples
Weighing 0.5g-1.0g of sample in a 25ml volumetric flask, adding about 5.0ml of water, performing vortex oscillation, fully dissolving the sample, adding 15.0ml of 60% methanol aqueous solution after dissolution, shaking up, performing ultrasonic extraction for 10min-15min, cooling the liquid to room temperature, fixing the volume to a scale by using the 60% methanol aqueous solution, shaking up, filtering by using an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times by using the 60% methanol aqueous solution, and detecting by using a high performance liquid chromatograph.
And 5: pretreatment of cake, bread and biscuit containing Bordeaux mixture
Crushing or shearing a sample by using a crusher, fully and uniformly stirring, weighing about 1.0g of sample in a beaker, adding 20ml of petroleum ether, uniformly stirring, placing for layering, pouring off a petroleum ether layer, repeating the operation for 2 times, fully removing fat in the sample, volatilizing the petroleum ether, adding about 20ml of 60% methanol aqueous solution, ultrasonically extracting for 30min, transferring the upper layer solution into a 25ml volumetric flask after the solution is cooled to room temperature, washing residues by using a small amount of 60% methanol aqueous solution, merging a washing solution into the volumetric flask, fixing the volume to scale by using the 60% methanol aqueous solution, shaking uniformly, and detecting by using a high performance liquid chromatograph.
Step 6: pretreatment of cosmetic containing Bordeaux mixture.
(1) Treatment of waxless samples (skin care type, shampoo type, powder type, etc.)
Weighing 2.5g of sample in a 10.0ml colorimetric tube, dissolving with 5.0ml of purified water, performing ultrasonic treatment for 30 minutes, cooling the temperature of a test solution to room temperature, metering the volume to a scale with 60% methanol water, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
(2) pretreatment of waxy samples (lipstick, etc.)
Weighing 2.5g of sample in a 10.0ml colorimetric tube, dissolving with 1ml of tetrahydrofuran, adding 4ml of methanol, vibrating on a vortex oscillator at high speed for 5min, carrying out ultrasonic treatment for 30min, cooling the temperature of the test solution to room temperature, metering the volume to scale with the methanol, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph.
Liquid phase conditions:
aqueous phase (a): high purity water; organic phase (B): acetonitrile; sample introduction conditions are as follows: injecting sample with 8% organic phase and 92% water phase at equal temperature for 15 min; the flow rate is 1.0 mL/min;
the column temperature is 25 ℃; the sample injection amount is 10 mu L.
The content of the active substance in the sample is calculated according to the formula (1):
in the formula:
x is the content of Borojo-iridoid glycoside in the sample, and the unit is milligram per kilogram and mg/100 g;
c, obtaining the concentration of the Borojo-iridoid glycoside in the tested sample solution from the standard working curve, wherein the unit is microgram per milliliter and microgram/mL;
n is the dilution multiple of the initial sample solution;
v is the constant volume of the initial sample liquid, and the unit is milliliter (mL);
m is the weight of the sample, in grams and g.
Note: the calculation result should be expressed to the last two decimal places.
Detection limit and quantification limit
The method has a concentration of 0.01 μ g/ml and a quantitative concentration of 0.03 μ g/ml, and when the sample volume is 1.0g and the constant volume is 10.0ml, the detection limit is 0.1mg/kg and the quantitative limit is 0.3mg/kg, and the high performance liquid chromatogram is shown in FIG. 4.
The test results of this example are shown in table 1 below:
TABLE 1 determination of the recovery of Borojo-iridoid glycoside by liquid chromatography
Example 2
Microanalysis method of Borojo-8, 9-iridoid glycoside as marker component in Borojo and its product (high performance liquid chromatography-mass spectrum internal standard method)
1. Reagents and solutions
The experimental water is first-grade water meeting the GB/T6682 specification.
Reagent: methanol (chromatographically pure); formic acid (chromatographically pure); acetonitrile (chromatographically pure); 8, 9-iridoid glycoside reference substance; internal standard substance: apigenin (China institute for testing and testing food and drug, purity > 99%, batch No.: 111901-201603).
Solution preparation:
1. standard solution
1.1 Standard stock solutions
10mg of the standard substance is accurately weighed and placed in a 10mL brown volumetric flask, and a stock solution with the concentration of 1.0mg/mL is prepared by using 60% methanol water.
The used stock solutions are protected from light by tinfoil and stored in a 4C refrigerator.
1.2 Standard use solutions
Standard stock solutions were prepared with water to a range of concentrations: 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.50, 25.00 and 50.00ng/mL standard solutions and a final concentration of 10ng.mL-1 apigenin internal standard solution was added.
2. Internal standard stock solution
10.0mg of the standard was accurately weighed, placed in a 10mL brown volumetric flask and prepared with water to give an internal standard stock solution having a concentration of 1.0 mg/mL.
2.1 internal standard use solution
The internal standard stock solution was diluted with water to a use solution concentration of 500 ng/ml.
2 instruments and apparatus
2.1 ultra-high performance liquid phase tandem mass spectrometer, model: us Waters, XEVOTQ-S, quaternary pump
2.2 ACQUICTIUPLC HSS T3 column, Specification: 2.1X100 mm,1.8um
2.3 refrigerated centrifuge, Eppendorf, Germany, Specifications: 5804R
2.4 electronic balance, model: BT-125D.
The experimental procedure for this example is as follows:
s1: pretreatment of Borojo raw fruit
Weighing 1.0g of sample into a beaker, adding 2.0mL of internal standard solution, adding 100mL of 60% methanol water, stirring uniformly, sealing the opening of the beaker by using tinfoil paper, heating in a water bath at 90 ℃ for 30 minutes, taking out the beaker, cooling to room temperature, centrifuging to obtain supernatant, filtering by using an ultrafiltration membrane of 0.22 mu m (or filtering by using filter paper, and then filtering the filtrate by using the ultrafiltration membrane), and detecting by using a high performance liquid chromatography-mass spectrometer.
S2: pretreatment of Borojo enzymolysis concentrated powder
Weighing 0.1g of sample into a beaker, adding 2.0ml of internal standard use solution, dissolving with 60% methanol to constant volume to 100ml, shaking up, filtering with 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer;
s3: pretreatment of Baole fruit wine
Weighing 1.0g of sample in a beaker, adding 0.5ml of internal standard solution, carrying out water bath in hot water at 60 ℃ for 20min, volatilizing ethanol, cooling the temperature of the sample solution to room temperature, then using 60% methanol water to fix the volume to 25ml, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
s4: pretreatment of beverage containing Bordeaux mixture
A: pretreatment of liquid samples
Weighing 1.0g of sample in a 25ml volumetric flask, adding 0.5ml of internal standard use solution, adding about 20ml of 60% methanol aqueous solution, carrying out ultrasonic extraction for 10min, cooling the liquid to room temperature, fixing the volume to the scale with the 60% methanol aqueous solution, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
b: pretreatment of solid samples
Weighing 0.5g-1.0g of sample in a 25ml volumetric flask, adding about 5.0ml of water, performing vortex oscillation, fully dissolving the sample, adding 0.5ml of internal standard use solution, adding 15.0ml of 60% methanol aqueous solution after dissolution, shaking up, performing ultrasonic extraction for 10min-15min, cooling the liquid to room temperature, metering to the scale with 60% methanol aqueous solution, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatograph.
S5: pretreatment of cake, bread and biscuit containing Bordeaux mixture
Crushing or shearing a sample by using a crusher, fully and uniformly stirring, weighing about 1.0g of sample in a beaker, adding 20ml of petroleum ether, uniformly stirring, standing for layering, pouring off a petroleum ether layer, repeating the operation for 2 times, fully removing fat in the sample, volatilizing the petroleum ether, adding about 20ml of 60% methanol aqueous solution, adding 0.5ml of internal standard solution, ultrasonically extracting for 30min, cooling the solution to room temperature, transferring the upper solution to a 25ml volumetric flask, washing residues by using a small amount of 60% methanol aqueous solution, merging a washing solution into the volumetric flask, fixing the volume to the scale by using the 60% methanol aqueous solution, shaking uniformly, and detecting by using a high performance liquid chromatograph;
s6: pretreatment of cosmetic containing Bordeaux mixture.
A: pretreatment of wax-free samples (skin care, shampoo, powder, etc.)
Weighing 2.5g of sample in a 10ml colorimetric tube, adding 0.2ml of internal standard use solution, dissolving with 4.0ml of purified water, carrying out ultrasonic treatment for 30 minutes, cooling the temperature of the test solution to room temperature, fixing the volume to the scale with 60% methanol water, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer;
b: pretreatment of waxy samples (lipstick, etc.)
Weighing 2.5g of sample into a 10ml colorimetric tube, adding 0.2ml of internal standard use solution, dissolving with 1ml of tetrahydrofuran, adding 4.0ml of methanol, vibrating on a vortex oscillator at high speed for 5min, carrying out ultrasonic treatment for 30min, cooling the temperature of the test solution to room temperature, fixing the volume to the scale with methanol, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer.
Liquid chromatography-mass spectrometry chromatography conditions: aqueous phase (a): 0.1% aqueous formic acid; organic phase (B): b, acetonitrile; sample introduction conditions are as follows: gradient elution; the flow rate is 0.3 mL/min; the column temperature is 25 ℃; the sample injection amount is 8 mu L; desolvation temperature: 350 ℃; taper hole voltage: 8V, and (2); collision voltage: 15V; taper hole gas flow: 30 fhr-1; desolventizing agent gas flow: 700L < hr-1; an ion source: ESI-mode; specific parameters of the multiple ion reaction monitoring channel (MRM) are shown in table 2.
TABLE 2 Borojo- -iridoid glycosides and MRM parameters of their internal standards
And selecting standard working solutions with similar peak areas according to the content of the Borojo-iridoid glycoside in the sample solution. The response value of the active matter components in the standard working solution and the sample solution is in the linear range of the detection of the instrument.
As a result, the content of Borojo-iridoid glycoside in the sample was calculated according to the formula (2):
in the formula:
x is the content of Borojo-iridoid glycoside in the sample, and the unit is milligram per kilogram and mg/100 g;
c, obtaining the concentration of the Borojo-iridoid glycoside in the tested sample solution from the standard working curve, wherein the unit is microgram per milliliter and microgram/mL;
n is the dilution multiple of the initial sample solution;
v is the constant volume of the initial sample liquid, and the unit is milliliter (mL);
m is the weight of the sample, in grams and g.
Detection limit and quantification limit: the method has the concentration of 5.0ng/ml and the quantitative concentration of 15.0ng/ml, when the sampling amount is 1.0g and the constant volume is 10.0ml, the detection limit is 0.05mg/kg and the quantitative limit is 0.15mg/kg, and the mass spectrometry scan is shown in figure 5.
The test results of this example are shown in table 3 below:
TABLE 1 determination of recovery of Borojo-iridoid glycoside by LC-MS
In order to overcome the defects of mass spectrum testing technology by an external standard method, the micro active substances in various different matrix tylophora products are tested, the interference of similar components in the products is eliminated, and the use of an internal standard is introduced. Meanwhile, a liquid chromatography analysis technology is established, and the accuracy of the detection result of the conventional sample is ensured.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (6)
1. The microanalysis method of the symbolic component Borojo-8, 9-iridoid glycoside in the Borojo and the products thereof is characterized by comprising the following steps:
step 1: pretreatment of Borojo raw fruit
Weighing 10g of sample into a beaker, adding 100mL of 60% methanol water, stirring uniformly, sealing the mouth of the beaker by using tinfoil paper, heating in a water bath at 90 ℃ for 30 minutes, taking out the beaker, cooling to room temperature, centrifuging to obtain supernatant, filtering by using an ultrafiltration membrane of 0.22 mu m, and detecting by using a high performance liquid chromatograph;
step 2: pretreatment of Borojo enzymolysis concentrated powder
Weighing 1.0g of sample into a beaker, dissolving with 60% methanol to a constant volume of 100ml, shaking up, filtering with an ultrafiltration membrane of 0.22 μm, diluting the filtrate 1000 times with 60% methanol water solution, and detecting with a high performance liquid chromatograph;
and step 3: pretreatment of Bordeaux fermented fruit wine
Weighing 1.0g of sample in a beaker, carrying out water bath in hot water at 60 ℃ for 20min, volatilizing ethanol in the sample, reducing the temperature of the sample liquid to room temperature, metering the volume to 25ml by using 60% methanol water solution, shaking up, filtering by using an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times by using 60% methanol water solution, and detecting by using a high performance liquid chromatograph;
and 4, step 4: pretreating the beverage containing the Bordeaux mixture;
and 5: pretreatment of cake, bread and biscuit containing Bordeaux mixture
Crushing or shearing a sample by using a crusher, fully and uniformly stirring, weighing about 1.0g of sample in a beaker, adding 20ml of petroleum ether, uniformly stirring, standing for layering, pouring off a petroleum ether layer, repeating the operation for 2 times, fully removing fat in the sample, volatilizing the petroleum ether, adding about 20ml of 60% methanol aqueous solution, ultrasonically extracting for 30min, transferring the upper layer solution into a 25ml volumetric flask after the solution is cooled to room temperature, washing residues by using a small amount of 60% methanol aqueous solution, merging a washing solution into the volumetric flask, fixing the volume to scale by using the 60% methanol aqueous solution, uniformly shaking, and detecting by using a high performance liquid chromatograph;
step 6: pretreatment of cosmetic containing Bordeaux mixture.
2. The microanalysis method of the marker ingredient Borojo-8, 9-iridoid glycoside of tylophora fruit and the products thereof as claimed in claim 1, wherein the step 4 comprises two parts of pretreatment of liquid sample and pretreatment of solid sample, wherein:
(1) pretreatment of liquid samples
Weighing 1.0g of sample in a 25ml volumetric flask, adding about 20ml of 60% methanol aqueous solution, carrying out ultrasonic extraction for 10min, cooling the liquid to room temperature, fixing the volume to the scale with the 60% methanol aqueous solution, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times with the 60% methanol aqueous solution, and detecting with a high performance liquid chromatograph;
(2) pretreatment of solid samples
Weighing 0.5g-1.0g of sample in a 25ml volumetric flask, adding about 5.0ml of water, performing vortex oscillation, fully dissolving the sample, adding about 15.0ml of 60% methanol aqueous solution after dissolution, shaking up, performing ultrasonic extraction for 10min-15min, cooling the liquid to room temperature, fixing the volume to a scale by using the 60% methanol aqueous solution, shaking up, filtering by using an ultrafiltration membrane of 0.22 mu m, diluting the filtrate by corresponding times by using the 60% methanol aqueous solution, and detecting by using a high performance liquid chromatograph.
3. The microanalysis method of the marker ingredient Borojo-8, 9-iridoid glycoside of tylophora fruit and its products as claimed in claim 1, wherein step 6 comprises two parts of pretreatment of non-waxy sample and pretreatment of waxy sample, wherein:
(1) pretreatment of non-waxy samples
Weighing 2.5g of sample in a 10.0ml colorimetric tube, dissolving with 5.0ml of purified water, performing ultrasonic treatment for 30 minutes, cooling the temperature of a test solution to room temperature, metering the volume to a scale with 60% methanol water, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
(2) pretreatment of waxy samples
Weighing 2.5g of sample in a 10.0ml colorimetric tube, dissolving with 1ml of tetrahydrofuran, adding 4ml of methanol, vibrating on a vortex oscillator at high speed for 5min, carrying out ultrasonic treatment for 30min, cooling the temperature of the test solution to room temperature, metering the volume to scale with the methanol, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph.
4. The microanalysis method of the marker ingredient Borojo-8, 9-iridoid glycoside of tylophora fruit and products thereof as claimed in claim 1, comprising the steps of:
s1: pretreatment of Borojo raw fruit
Weighing 1.0g of sample into a beaker, adding 2.0mL of internal standard solution, adding 100mL of 60% methanol water, stirring uniformly, sealing the opening of the beaker by using tinfoil paper, heating in a water bath at 90 ℃ for 30 minutes, taking out the beaker, cooling to room temperature, centrifuging to obtain supernatant, filtering by using an ultrafiltration membrane of 0.22 mu m, and detecting by using a high performance liquid chromatography-mass spectrometer;
s2: pretreatment of Borojo enzymolysis concentrated powder
Weighing 0.1g of sample into a beaker, adding 2.0ml of internal standard use solution, dissolving with 60% methanol to constant volume to 100ml, shaking up, filtering with 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer;
s3: pretreatment of Bordeaux fermented fruit wine
Weighing 1.0g of sample in a beaker, adding 0.5ml of internal standard solution, carrying out water bath in hot water at 60 ℃ for 20min, volatilizing ethanol, cooling the temperature of the sample solution to room temperature, then using 60% methanol water to fix the volume to 25ml, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
s4: pretreatment of beverage containing Bordeaux mixture
S5: pretreatment of cake, bread and biscuit containing Bordeaux mixture
Crushing or shearing a sample by using a crusher, fully and uniformly stirring, weighing about 1.0g of sample in a beaker, adding 20ml of petroleum ether, uniformly stirring, standing for layering, pouring off a petroleum ether layer, repeating the operation for 2 times, fully removing fat in the sample, volatilizing the petroleum ether, adding about 20ml of 60% methanol aqueous solution, adding 0.5ml of internal standard solution, ultrasonically extracting for 30min, cooling the solution to room temperature, transferring the upper solution to a 25ml volumetric flask, washing residues by using a small amount of 60% methanol aqueous solution, merging a washing solution into the volumetric flask, fixing the volume to the scale by using the 60% methanol aqueous solution, shaking uniformly, and detecting by using a high performance liquid chromatograph;
s6: pretreatment of cosmetic containing Bordeaux mixture.
5. The microanalysis method of the marker ingredient Borojo-8, 9-iridoid glycoside of tylophora fruit and products thereof as set forth in claim 4, wherein S4 includes two parts of liquid sample treatment and solid sample treatment, wherein:
a: pretreatment of liquid samples
Weighing 1.0g of sample in a 25ml volumetric flask, adding 0.5ml of internal standard use solution, adding about 20ml of 60% methanol aqueous solution, carrying out ultrasonic extraction for 10min, cooling the liquid to room temperature, fixing the volume to the scale with the 60% methanol aqueous solution, shaking up, filtering with an ultrafiltration membrane of 0.22 mu m, and detecting with a high performance liquid chromatograph;
b: pretreatment of solid samples
Weighing 0.5g-1.0g of sample in a 25ml volumetric flask, adding about 5.0ml of water, performing vortex oscillation, fully dissolving the sample, adding 0.5ml of internal standard use solution, adding 15.0ml of 60% methanol aqueous solution after dissolution, shaking up, performing ultrasonic extraction for 10min-15min, cooling the liquid to room temperature, metering to the scale with 60% methanol aqueous solution, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatograph.
6. The microanalysis method of the marker ingredient Borojo-8, 9-iridoid glycoside of tylophora fruit and its products as claimed in claim 4, wherein S6 includes pretreatment of non-waxy sample and pretreatment of waxy sample, wherein:
a: treatment of wax-free samples
Weighing 2.5g of sample in a 10ml colorimetric tube, adding 0.2ml of internal standard use solution, dissolving with 4.0ml of purified water, carrying out ultrasonic treatment for 30 minutes, cooling the temperature of the test solution to room temperature, fixing the volume to the scale with 60% methanol water, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer;
b: treatment of waxy samples
Weighing 2.5g of sample into a 10ml colorimetric tube, adding 0.2ml of internal standard use solution, dissolving with 1ml of tetrahydrofuran, adding 4.0ml of methanol, vibrating on a vortex oscillator at high speed for 5min, carrying out ultrasonic treatment for 30min, cooling the temperature of the test solution to room temperature, fixing the volume to the scale with methanol, shaking up, filtering with a 0.22 mu m ultrafiltration membrane, and detecting with a high performance liquid chromatography-mass spectrometer.
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