CN113567578A - SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar - Google Patents
SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar Download PDFInfo
- Publication number
- CN113567578A CN113567578A CN202110802713.XA CN202110802713A CN113567578A CN 113567578 A CN113567578 A CN 113567578A CN 202110802713 A CN202110802713 A CN 202110802713A CN 113567578 A CN113567578 A CN 113567578A
- Authority
- CN
- China
- Prior art keywords
- des
- catechin
- vinegar
- epicatechin
- flavanols
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- 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
-
- 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
- G01N30/08—Preparation using an enricher
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Pyrane Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of flavanol substance detection, and discloses an SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar. The method adopts a Diamonsil C18 chromatographic column, and the flow rate is 1 mL/min; the sample injection amount is 10 mu L; the detection wavelength is 280 nm; the mobile phase is 0.1% phosphate buffer and acetonitrile; gradient elution. Optimization experiments finally determine that the amount of the XAD-2 macroporous adsorption resin after activation is 188mg, the volume of a eutectic solvent (tetraethylammonium chloride-n-octanoic acid, the molar ratio is 1:3) is 400 mu L, the adsorption time is 11min, and the enrichment effect of the two flavanols is optimal when the resolution time is 20 min. Under these conditions, the linearity of catechin and epicatechinThe relationship is good (R)2>0.99), detection limit, quantitative limit, relative standard deviation in day and standard recovery rate show that compared with the traditional complex solid phase extraction process, the method is simpler, more convenient, high in sensitivity and small in matrix interference, and is suitable for enriching and determining catechin and epicatechin in Shanxi mature vinegar.
Description
Technical Field
The invention belongs to the technical field of flavanol substance detection, and particularly relates to an SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar.
Background
Catechin and epicatechin are active flavanols mainly present in natural plants such as tea leaves. Researches show that catechin has the effects of resisting oxidation, inflammation, obesity, bacteria and virus, relieving bone loss, preventing food poisoning, promoting the absorption of active substances in intestinal tracts, reducing the infection rate of influenza, preventing oral diseases such as dental caries and the like, preventing cardiovascular diseases and cancers, protecting erythrocytes from permanganate-induced hemolysis, oxidation of erythrocyte protein sulfydryl and membrane lipid peroxidation, and even preventing and treating neurodegenerative diseases such as Alzheimer's disease and the like. Catechin can be rapidly metabolized after entering the body of mammal through the conventional way, and the substantial chemical modification of catechin to play a role is a direction of future research, and the potential in the aspect of drug design is yet to be developed. In addition, (+) -catechin can be used as a coloring agent of a high molecular material and an indicator of aging time, and has wide application prospect in the packaging industry. Epicatechin has good effects in resisting cancer, treating diabetes, resisting inflammation, oxidation, cardiovascular diseases, protecting nerve, and promoting muscle growth. Cancer and diabetes have serious effects on human life, and the intake of epicatechin has been shown to lower blood glucose levels in diabetic patients, and the anticancer effects are attributed to its antioxidant properties, anti-angiogenesis, and cytotoxicity directly to cancer cells.
As one of four major Chinese vinegars, Shanxi mature vinegar is made from five cereals such as high-quality sorghum, barley, peas and the like through steaming, fermenting, fumigating, showering and drying in the sun, and contains various components such as rich amino acid, organic acid, sugar, polyphenol, flavone, vitamin and the like due to the unique brewing process. As a flavoring agent, vinegar also has health promotion effects of stimulating appetite, promoting digestion, relieving fatigue, lowering blood pressure, reducing blood sugar, reducing blood lipid, etc. Research reports that the Shanxi mature vinegar contains catechin and epicatechin, but the Shanxi mature vinegar has complex components, and trace active ingredients cannot be directly measured due to interference of a matrix. The direct enrichment of trace active ingredients in the mature vinegar, and the selection of a proper eluent to elute the active ingredients after the enrichment is a key factor to be solved, but there are only few literature reports on the determination of the content of catechin and epicatechin in Shanxi mature vinegar at present.
Based on the method, the invention aims to establish a method for rapidly and efficiently measuring catechin and epicatechin in Shanxi mature vinegar by combining solid-phase extraction with eutectic solvent-high performance liquid chromatography. By quickly detecting catechin and epicatechin in the vinegar sample, a theoretical basis is provided for the content of the flavonols in the brewed vinegar.
Disclosure of Invention
Aiming at the problems, the invention provides an SPE-DES-HPLC method for simultaneously measuring two flavanol substances in Shanxi mature vinegar. (ii) a
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an SPE-DES-HPLC method for simultaneously measuring two flavanol substances in Shanxi mature vinegar, which is characterized by comprising the following steps of: the method comprises the following steps:
the chromatographic column is a Diamonsil C18 chromatographic column with the diameter of 4.6mm multiplied by 250mm and the diameter of 5 mu m; the detector is an ultraviolet detector; the flow rate is 1 mL/min; the sample injection amount is 10 mu L; the column temperature was 35 ℃; the detection wavelength is 280 nm; the mobile phase is phosphate buffer solution and acetonitrile with the volume percentage of 0.1 percent; the elution mode is gradient elution, and is specifically shown in the following table:
Putting 1g of macroporous resin into a container, adding 5ml of absolute ethyl alcohol for activation for 2 hours, and washing with distilled water until no alcohol smell exists, thereby completing resin activation;
Standard stock solutions: accurately weighing 10mg catechin and epicatechin standard substances respectively, and fixing the volume to 10mL by using chromatographic grade methanol to obtain mother liquor with the concentration of 1 mg/mL;
standard working solution: weighing 500 μ L of catechin and epicatechin mother liquor, and diluting to 10mL with methanol to obtain mixed standard solution with catechin and epicatechin mass concentration of 50 μ g/mL; diluting with chromatographic grade methanol to obtain mixed standard solutions with mass concentrations of 0.1, 0.2, 0.5, 5, 10, 20 and 50 μ g/mL respectively;
simulating a vinegar sample: measuring the pH value of Shanxi mature vinegar to be 3.42, so that 50mL of acetic acid solution with the pH value of 3.42 is prepared; accurately transferring 500 mu L of 50 mu g/mL mixed standard solution, adding 2.00mL of acetic acid solution with pH of 3.42 to prepare a simulated vinegar sample;
Adsorption: taking 2.5mL of simulated vinegar sample and 100 mg of XAD-2 macroporous adsorption resin activated in the step 2, and adsorbing for 5-25 min;
and (3) analysis: after adsorption, centrifuging at 6000rpm for 5min, discarding the supernatant, adding 200 and 600 μ L of eluent, and performing vortex analysis for 5-25 min; centrifuging at 6000rpm for 5min, collecting supernatant, filtering with 0.45 μm organic filter membrane, and performing high performance liquid chromatography;
Pretreating the mixed standard solution with each concentration prepared in the step 3 according to the method in the step 4 in sequence, detecting by adopting the high performance liquid chromatography condition determined in the step 1, determining by retention time, drawing a standard curve according to the corresponding relation between the peak area of each concentration and the concentration of the peak area, respectively determining the linear regression equation of the two flavanols,
the linear regression equation of the two flavanols is as follows:
catechin: 26.747x-9.338, R20.9917, x applies: 0.5-50 mug/mL;
epicatechin: 73.919x-37.145, R20.9928, x applies: 0.2-50 mug/mL;
wherein: y is the peak area corresponding to flavanols, x is the mass concentration of flavanols, R2Is a linear correlation coefficient;
Diluting 5 actual vinegar samples by 10-20 times with distilled water, pretreating according to the method in the step 4, detecting by adopting the high performance liquid chromatography condition determined in the step 1, measuring peak areas of components in the samples, determining the qualitative by retention time, and calculating the contents of two flavanol substances, namely catechin and epicatechin in the actual vinegar samples according to the linear regression equation determined in the step 5.
Further, the eluent in the step 4 comprises: DES with molar ratio of anhydrous ethanol, 70% ethanol, ethyl acetate, methanol, tetraethyl ammonium chloride and n-octanoic acid of 1:2-51DES with 1:2 molar ratio of tetrabutylammonium chloride to caprylic acid2And the molar ratio of choline chloride to acetic acid is 1:2 DES3Preferably DES with a 1:3 molar ratio of tetraethylammonium chloride to caprylic acid1。
The optimal dosage of the XAD-2 macroporous adsorption resin activated in the step 4 is 188mg, and the optimal adsorption time is 11 min.
The optimal dosage of the eluent in the step 4 is 400 mu L, and the optimal resolution time is 20 min.
Compared with the prior art, the invention has the following advantages:
1. the invention establishes a rapid high-efficiency detection method combining solid-phase extraction with eutectic solvent-high performance liquid chromatography, and the specific chromatographic conditions are as follows: a Diamonsil C18 chromatographic column with the size of 4.6mm multiplied by 250mm and the size of 5 mu m is selected; an ultraviolet detector; the flow rate is 1 mL/min; the sample injection amount is 10 mu L; the column temperature was 35 ℃; the detection wavelength is 280 nm; the mobile phase is phosphate buffer solution and acetonitrile with the volume percentage of 0.1 percent; the elution mode is gradient elution; the eluent for the pretreatment of the sample is tetraethylammonium chloride and normalDES with octanoic acid molar ratio of 1:31400 mul, 188mg of activated XAD-2 macroporous adsorption resin and 11min of optimal adsorption time; the optimal resolution time is 20 min.
2. Linear regression equation R for the method of the invention2The standard addition recovery rate is more than or equal to 0.99, the detection Limit (LOD) is 0.1-0.2, the quantification Limit (LOQ) is 0.2-0.5, the enrichment times are all more than 30, the relative standard deviation in the day is 0.3-0.97, the relative standard deviation in the day is 0.96-4.26, and the standard addition recovery rate is 98.8-118.8%. The method has the advantages of good precision and accuracy, high sensitivity and good enrichment effect. The process of the invention is also used. The retention times of catechin and epicatechin were 8min and 10min, respectively, indicating that complete separation of the two flavanols could be achieved under the present method.
3. Traditional solid phase extraction generally needs to purchase the solid phase extraction post of commercialization, needs a certain amount of solvent activation before using, and sample solution after the activation crosses the post and adsorbs, uses the impurity in the solvent elution sample, and the solvent elution enrichment target object of different polarity of reuse, the solvent volume that needs when eluting the target object is great, and the great eluant of volume needs nitrogen to weather, and the residual is solvent redissolved once more, and the process is comparatively loaded down with trivial details. The method combines solid phase extraction, the eutectic solvent and high performance liquid chromatography, and compared with the traditional complex solid phase extraction process, the method is simple, rapid, high in sensitivity, small in matrix interference and high in enrichment factor, the eutectic solvent used in the method has the advantages of easiness in preparation, low melting point, low toxicity, low cost and the like, can be used for rapid detection of flavonols in vinegar, and provides a new direction and thought for subsequent research.
Drawings
FIG. 1 shows the effect of different elution solvents on the analysis of catechin and epicatechin.
FIG. 2 is a graph showing the effect of different amounts of activated XAD-2 macroporous resin on the solid phase extraction of catechin and epicatechin.
FIG. 3 DES with different molar ratios1Influence on enrichment effect of catechin and epicatechin.
FIG. 4 shows DES of different volumes1Influence on the extraction efficiency of catechin and epicatechin.
FIG. 5 is a graph showing the effect of adsorption time on the extraction efficiency of catechin and epicatechin.
FIG. 6 is a graph showing the effect of analysis time on the extraction efficiency of catechin and epicatechin.
FIG. 7 is a Pareto chart of the factors of two flavanols of the present invention (upper panel is catechin, lower panel is epicatechin), wherein A: DES (data encryption Standard)1A molar ratio; b: DES (data encryption Standard)1Volume: c: the dosage of the activated XAD-2 macroporous resin; d: adsorption time; e: and (5) analyzing time.
FIG. 8 shows DES1Ratio (X)1) And the dosage of the activated XAD-2 macroporous adsorption resin (X)2) Adsorption time (X)3) Response surface graph and contour line analysis graph of catechin in Shanxi mature vinegar.
FIG. 9 shows DES1Ratio (X)1) And the dosage of the activated XAD-2 macroporous adsorption resin (X)2) Adsorption time (X)3) Response surface graph and contour line analysis graph of epicatechin in Shanxi mature vinegar.
FIG. 10 is a chromatogram of two flavanols in a standard solution, diluted vinegar-like V-4. Wherein A is the chromatogram of the standard solution, and B is the chromatogram of the diluted vinegar sample V-4.
Detailed Description
The technical solution in the embodiments of the present invention will be specifically and specifically described below with reference to the embodiments of the present invention and the accompanying drawings. It should be noted that variations and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.
5 Shanxi mature vinegar samples (numbered as V-1, V-2, V-3, V-4 and V-5) in the following examples were purchased from supermarkets; XAD-2 macroporous adsorbent resin (Nanjing Tolye Biotechnology Co., Ltd.); tetraethylammonium chloride, tetrabutylammonium chloride, choline chloride, n-decanoic acid, n-octanoic acid (Shanghai Michelin Biochemical technology Co., Ltd.); glacial acetic acid (grand borrelid chemical technologies, ltd); phosphoric acid (Sichuan Andan nongsen science and technology Co., Ltd.); catechin standards, epicatechin standards (shanghai, e.g., ji biotech development ltd); methanol, acetonitrile (chromatographically pure, bruke corporation); 5mL centrifuge tubes (Nantong Chunming laboratory instruments Co., Ltd.); 0.45 μm organic filter (Jinteng laboratory Co., Tianjin).
Agilent1260 Infinity II high performance liquid chromatograph (Agilent Inc., USA); AL204 electronic analytical balance (digraph (shanghai) biotechnology limited); TG16A-W high speed centrifuge (Hunan Saite Xiang instrumental Co., Ltd.); MX-S adjustable mixer (DLAB Beijing Dalong instrument); PB-10 acidimeters (Shanghai Bo instruments Co., Ltd.); solvent filters (Zhejiang Naddy scientific instruments, Inc.); a circulating water type multipurpose vacuum pump (west amp mogina instrument manufacturing ltd).
Example 1: experimental method for simultaneously determining two flavanols in Shanxi mature vinegar
1. High performance liquid chromatography conditions
Agilent1260 Infinity II high performance liquid chromatograph; the chromatographic column is a Diamonsil C18 chromatographic column with the diameter of 4.6mm multiplied by 250mm and the diameter of 5 mu m; the detector is an ultraviolet detector; the flow rate is 1 mL/min; the sample injection amount is 10 mu L; the column temperature was 35 ℃; the detection wavelength is 280 nm; the mobile phase is phosphate buffer solution and acetonitrile with the volume ratio of 0.1 percent; the elution mode is gradient elution, which is specifically shown in the following table 1:
TABLE 1 gradient elution procedure
2. Activation of XAD-2 macroporous adsorbent resin
Putting 1g of macroporous resin into a beaker, adding 5ml of absolute ethyl alcohol for activation for 2 hours, and washing with distilled water until no alcohol smell exists, thereby completing the resin activation;
3. preparation of standard stock solution, standard working solution and simulated vinegar sample
Standard stock solutions: accurately weighing 10mg catechin and epicatechin standard substances respectively, and performing constant volume in a 10mL volumetric flask by using chromatographic grade methanol to obtain a mother solution with the concentration of 1 mg/mL;
standard working solution: weighing 500 μ L of catechin and epicatechin mother liquor in volumetric flasks, and diluting to 10mL with methanol to obtain mixed standard solution with catechin and epicatechin mass concentrations of 50 μ g/mL; diluting with chromatographic grade methanol to obtain mixed standard solutions with mass concentrations of 0.1, 0.2, 0.5, 5, 10, 20 and 50 μ g/mL respectively;
simulating a vinegar sample: the pH value of Shanxi mature vinegar is 3.42 measured by a PB-10 acidimeter, so that 50mL of acetic acid solution with the pH value of 3.42 is prepared; accurately transferring 500 mu L of 50 mu g/mL mixed standard solution, adding 2.00mL of acetic acid solution with pH of 3.42 to prepare a simulated vinegar sample;
4. pretreatment of vinegar sample
Adding 2.5mL of simulated vinegar sample and 188mg of XAD-2 macroporous adsorption resin activated in the step 2 into a 5mL centrifuge tube, and vortexing for 11min to achieve sufficient adsorption; after centrifugation at 6000rpm for 5min, the supernatant solution was discarded, 400. mu.L of eluent was added and vortexed for 20min to achieve full resolution. Centrifuging again, collecting supernatant, filtering with 0.45 μm organic filter membrane, and performing high performance liquid chromatography.
Example 2: optimization of the Experimental conditions
One, one factor test optimization
1. Selection of elution solvent
Since different eluents have different acting forces with the target analyte, the kind of elution solvent has a large influence on the solid phase extraction efficiency. Thus, the present invention selects eluents: anhydrous ethanol, 70% ethanol, ethyl acetate, methanol, DES1(tetraethylammonium chloride: n-octanoic acid 1:2, molar ratio), DES2(tetrabutylammonium chloride: n-octanoic acid ═ 1:2, molar ratio), DES3(Choline chloride: acetic acid ═ 1:2, molar ratio), the effect of the compound on the analysis effect of catechin and epicatechin was examined by the experimental method described in example 1.
As can be seen from fig. 1, the elution effects of the seven elution solvents on catechin were ranked as follows: ethyl acetate>DES1>Anhydrous ethanol>Methanol>70% ethanol>DES3>DES2(ii) a The elution effect on epicatechin was ranked as: DES (data encryption Standard)1>Anhydrous ethanol>Methanol>70% ethanol>Ethyl acetate>DES3>DES2. In other words, ethyl acetate has a good elution effect on catechins, DES1The elution of epicatechin was good, however, DES was found to be effective1The elution effect on two flavanols is excellent, so DES is selected in the invention1As the optimum elution solvent for this experiment.
2. Influence of amount of activated XAD-2 macroporous adsorbent resin on extraction efficiency of catechin and epicatechin
The invention selects 100-500mg of activated XAD-2 macroporous adsorption resin, and adopts the experimental method described in example 1 (wherein, the eluent is DES with the molar ratio of tetraethylammonium chloride to n-octanoic acid being 1:21) The influence of the amount of the activated XAD-2 macroporous adsorbent resin on the extraction efficiency of catechin and epicatechin was studied.
As can be seen from the analysis of FIG. 2, when the amount of the macroporous adsorbent resin is 100-200mg, the extraction efficiency of catechin and epicatechin is significantly improved along with the increase of the amount of the resin; when the amount of the macroporous adsorption resin is 200-500mg, the extraction efficiency of catechin and epicatechin is obviously reduced with the increase of the amount of the resin, which is probably because the excessive adsorbent causes incomplete elution of the target compound, thereby reducing the solid phase extraction efficiency. Thus, the final selected amount of activated XAD-2 macroporous resin of the present invention was selected to be 200 mg.
3、DES1Selection of the molar ratio
Determining DES1After optimizing the elution solvent for this experiment, the present inventors have further investigated DES1Influence of the molar ratio of tetraethylammonium chloride to n-octanoic acid (1:2-5) on the enrichment effect of catechin and epicatechin. The experimental method was finalized for example 1 combining steps 1 and 2.
As can be seen from FIG. 3, with DES1The enrichment effect of epicatechin gradually decreases with the increase of the molar ratio. Therefore, when the molar ratio of the tetraethylammonium chloride to the n-octanoic acid is 1:2, the enrichment effect of the epicatechin is the best; when DES1The catechin enrichment effect gradually increases with the increase of the molar ratio when the molar ratio is 1:2-1:3, and when DES is used1The molar ratio is 1:3-1:at 5, the effect of catechin enrichment is not greatly affected by the increase of the molar ratio. Therefore DES1When the molar ratio is 1:3, the best extraction effect of the catechin can be achieved. Taken together, the invention selects DES1The molar ratio of the tetraethylammonium chloride to the octanoic acid is 1: 3.
4、DES1Selection of volume
It has been shown that the amount of eluent can affect the elution degree of the target analyte, thereby having a great influence on the extraction effect. Therefore, the method used in step 3 is combined with the DES finally determined in step 31DES with molar ratio study volume of 200-1Influence on the extraction efficiency of catechin and epicatechin.
As can be seen from FIG. 4, when DES is used1The extraction efficiency of catechin and epicatechin is determined by DES when the volume is 200-1The volume is increased and obviously increased; after 400 μ L, DES1The volume is continuously increased, so that the extraction effect is not greatly influenced, and the DES is selected by the invention1The optimal volume of (2) is 400. mu.L.
5. Selection of adsorption time
DES finally determined by combining the experimental method used in step 4 with step 41The effect of adsorption time of 5-25min on the extraction of catechins and epicatechins was investigated by volume.
As can be seen from FIG. 5, when the adsorption time was 5-10min, the extraction effect of catechin and epicatechin increased significantly with the increase of time; after 10min, the extraction effect generally appears to be in a downward trend along with the increase of time, so the optimal adsorption time is 10 min.
6. Selection of resolution time
The effect of the resolving time of 5-25min on the extraction effect of catechin and epicatechin was investigated in combination with the adsorption time finally determined in step 5 by the experimental method used in step 5.
As can be seen from FIG. 6, the extraction efficiency of catechin gradually increased with the time lapse within 5-25 min; within 5-20min, the extraction efficiency of epicatechin gradually increases along with the prolonging of the resolution time. After 20min, the extraction efficiency gradually decreased with the longer resolving time, which may be caused by re-adsorption of the resolved target compound by the resin. Therefore, the present invention selects 20min as the best analysis time.
Second, Plackett-Burman test (selection of optimal combination)
The Plackett-Burman design is a design method for rapidly and effectively screening the most important factors from a plurality of variables by utilizing a first-order polynomial equation. The invention designs 12 groups of experiments by using software Design expert 8.0.6 to carry out DES1Molar ratio (A), DES1The significance of volume (B), amount of activated XAD-2 macroporous adsorbent resin (C), adsorption time (D) and analysis time (E) was examined. Each factor was set to two levels, high and low, as shown in table 2. Plackett-Burman design and results are shown in Table 3.
TABLE 2 variables and levels of Plackett-Burman test
TABLE 3Plackett-Burman test design and results
The Pareto plots clearly reflect the orderly normalization effect of each parameter, which in turn identifies the importance of each parameter, as long as the normalization effect of each parameter exceeds the vertical line in the Pareto plots (t-value <0.05), which represents statistical significance at the 95% confidence level.
FIG. 7 shows that DES1The mole ratio (A) and the dosage (C) of the activated XAD-2 macroporous adsorption resin are main significant influencing factors of catechin; DES (data encryption Standard)1The mole ratio (A), the dosage (C) of the activated XAD-2 macroporous adsorption resin and the adsorption time (D) are main significant influencing factors of epicatechin. Thus, the present invention selects DES globally1The molar ratio (A) of the activated XAD-2 is largerThe dosage (C) and adsorption time (D) of the porous adsorption resin are the main significant factors of two flavanols, wherein DES (DES)1The influence of the molar ratio (A) is most obvious, and the influence of the dosage (C) of the activated XAD-2 macroporous adsorption resin and the adsorption time (D) is second. Based on Plackett-Burman test results, non-significant factors were fixed as: DES (data encryption Standard)1The volume is 400 mu L, the analysis time is 20min, and the extraction condition is continuously optimized.
Third, response surface experiment
The response surface method is a statistical method utilizing the functional relationship between multivariate quadratic equation simulation factors and response values, is convenient, fast, good in repeatability and high in accuracy, and can be used for analyzing experiments by using a prediction model. The invention takes the P1ackett-Burman test result as the basis, and 3 factors with obvious influence are selected: DES (data encryption Standard)1Molar ratio (X)1) And the dosage of the activated XAD-2 macroporous adsorption resin (X)2) Adsorption time (X)3) A response surface Box-Behnken (BBD) test is designed, and extraction conditions are optimized. Design-Expert 8.0.6 software was used to Design a total of 17 trials at 3 factor 3 levels, with three parallel averaging sets for each trial. The design and results are shown in Table 4.
TABLE 4 response surface test and results
The quadratic multiple regression equation for two flavanols is as follows: the catechin is as follows: y is1＝271.16-12.84A-8.49B+1.55C+3.72AB-5.95AC+2.20BC-19.39A2-43.44B2-14.07C2The epicatechin is: y is2＝143.80-7.96A-3.45B+1.61C+1.50AB+0.93AC-2.55BC-5.49A2-17.56B2-4.14C2. A, B, C in the equation represent DES respectively1Proportion, dosage of the activated XAD-2 macroporous adsorption resin and adsorption time. Correlation coefficients of quadratic multiple regression equationIs R2 Catechin＝0.9832、R2 Epimetechin＝0.9469，R2 Adj Catechin＝0.9617、R2 adj epicatechin0.8785, indicating that the equation has a better fit. F and P values of the model are respectively FCatechin＝45.65，PCatechin<0.0001(FEpimetechin＝13.86，PEpimetechin0.0011), the mismatching term is not significant (P)Catechin＝0.9183>0.05；PEpimetechin＝0.1237>0.05), which means the model is significant.
In the case of catechins, DES was determined by anova1Molar ratio (X)1) And the dosage of the activated XAD-2 macroporous adsorption resin (X)2) Adsorption time (X)3) The F values of (a) and (b) were 40.52, 17.71, and 0.59, respectively, and the magnitude order of the effect of each factor on catechin extraction was: DES (data encryption Standard)1Molar ratio (X)1)>The dosage of the activated XAD-2 macroporous adsorbent resin (X)2)>Adsorption time (X)3) The test result is consistent with the test result of P1 ackett-Burman.
DES1Molar ratio (X)1) And the dosage of the activated XAD-2 macroporous adsorption resin (X)2) Adsorption time (X)3) The influence of three factors on the extraction efficiency of the target compounds (catechin and epicatechin) and the interaction and contour lines among the factors are shown in fig. 8-9. The results show that the optimal conditions for extracting the flavonols in the vinegar are as follows: DES (data encryption Standard)1Molar ratio (X)1) The dosage of the activated XAD-2 macroporous adsorption resin is 2.50 (X)2) 187.73mg, adsorption time (X)3) It is 10.81 min. Considering the feasibility of the actual operation, the conditions were modified as follows: DES (data encryption Standard)1Molar ratio (X)1) The dosage of the activated XAD-2 macroporous adsorption resin (X) is 32) 188mg, adsorption time (X)3) It is 11 min.
Thus, DES is finally determined1DES with a ratio of 31The volume is 400 μ L, the resolving time is 20min, the dosage of the activated XAD-2 macroporous adsorption resin is 188mg, and the adsorption time is 11 min.
EXAMPLE 3 actual Vinegar-like assay
1. Establishing a standard curve regression equation of catechin and epicatechin, detecting limit LOD (signal to noise ratio >3), quantifying limit LOQ (signal to noise ratio >10), and measuring enrichment times and relative standard deviation in the daytime and the day;
the formula for calculating the enrichment factor is as follows:
in the formula, CsedRepresents the concentration of the target in the eutectic; c0: initial target concentration
Pre-treating the mixed standard solution with each concentration prepared in example 1 according to the conditions determined in example 2, detecting by using the high performance liquid chromatography conditions of example 1, determining by retention time, drawing a standard curve according to the corresponding relation between the peak area size and the concentration of each concentration, and determining the linear regression equation, the linear range and the linear correlation coefficient (R) of the two flavanols respectively2). The results are shown in Table 5.
Standard Curve, detection Limit, quantitation Limit, relative Standard deviation between days for epicatechin 5 and epicatechin
From Table 5, R of the linear regression equation of the present invention can be obtained2The concentration is more than or equal to 0.99, the detection Limit (LOD) is 0.1-0.2, the quantification Limit (LOQ) is 0.2-0.5, the enrichment times are all more than 30, the relative standard deviation in the day is 0.3-0.97, and the relative standard deviation in the day is 0.96-4.26. The method has the advantages of good precision, high sensitivity and good enrichment effect.
2. Precision and recovery from spiked samples
The spiked samples (4, 8, 25. mu.g/mL) were pretreated and then examined by the pretreatment method described in example 1.
Results of standard recovery test of epicatechin 6 and epicatechin
As can be seen from Table 6, the recovery rates of the two products are 98.8% -118.8%, which indicates that the method has high precision and good accuracy.
3. Detection of content of actual acephate theanine and epicatechin
5 Shanxi mature vinegar were purchased from a supermarket and numbered V-1, V-2, V-3, V-4 and V-5 respectively. In order to reduce the substrate interference, V-1, V-2 and V-3 mature vinegar was diluted 10 times with distilled water, and V-4 and V-5 mature vinegar was diluted 20 times, and then the contents of catechin and epicatechin of 5 Shanxi mature vinegar samples were measured according to the conditions finally determined in examples 1 and 2, and the results are shown in FIG. 10 and Table 7.
Detection results of two flavanols in Table 75 Shanxi mature vinegar
FIG. 10A is chromatogram of standard solution of catechin and epicatechin, FIG. 10B is chromatogram of diluted vinegar sample (V-4), from which it can be seen that retention time of catechin is 8min and retention time of epicatechin is 10min, indicating that two flavanols can be completely separated under the experimental conditions of the present invention.
As can be seen from Table 7, the catechin content in the 5 vinegar samples ranged from 0.0544-1.0592 mg/mL, and the epicatechin content ranged from 0.0067-0.3360 mg/mL.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (4)
1. An SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar is characterized in that: the method comprises the following steps:
step 1, determining high performance liquid chromatography conditions:
the chromatographic column is a Diamonsil C18 chromatographic column with the diameter of 4.6mm multiplied by 250mm and the diameter of 5 mu m; the detector is an ultraviolet detector; the flow rate is 1 mL/min; the sample injection amount is 10 mu L; the column temperature was 35 ℃; the detection wavelength is 280 nm; the mobile phase is phosphate buffer solution and acetonitrile with the volume percentage of 0.1 percent; the elution mode is gradient elution, and is specifically shown in the following table:
step 2, activation of XAD-2 macroporous adsorbent resin
Putting 1g of macroporous resin into a container, adding 5ml of absolute ethyl alcohol for activation for 2 hours, and washing with distilled water until no alcohol smell exists, thereby completing resin activation;
step 3, preparing standard stock solution, standard working solution and simulated vinegar sample
Standard stock solutions: accurately weighing 10mg catechin and epicatechin standard substances respectively, and fixing the volume to 10mL by using chromatographic grade methanol to obtain mother liquor with the concentration of 1 mg/mL;
standard working solution: weighing 500 μ L of catechin and epicatechin mother liquor, and diluting to 10mL with methanol to obtain mixed standard solution with catechin and epicatechin mass concentration of 50 μ g/mL; diluting with chromatographic grade methanol to obtain mixed standard solutions with mass concentrations of 0.1, 0.2, 0.5, 5, 10, 20 and 50 μ g/mL respectively;
simulating a vinegar sample: measuring the pH value of Shanxi mature vinegar to be 3.42, so that 50mL of acetic acid solution with the pH value of 3.42 is prepared; accurately transferring 500 mu L of 50 mu g/mL mixed standard solution, adding 2.00mL of acetic acid solution with pH of 3.42 to prepare a simulated vinegar sample;
step 4, pretreatment of vinegar sample
Adsorption: taking 2.5mL of simulated vinegar sample and 100 mg of XAD-2 macroporous adsorption resin activated in the step 2, and adsorbing for 5-25 min;
and (3) analysis: after adsorption, centrifuging at 6000rpm for 5min, discarding the supernatant, adding 200 and 600 μ L of eluent, and performing vortex analysis for 5-25 min; centrifuging at 6000rpm for 5min, collecting supernatant, filtering with 0.45 μm organic filter membrane, and performing high performance liquid chromatography;
step 5, establishing a standard curve
Pretreating the mixed standard solution with each concentration prepared in the step 3 according to the method in the step 4 in sequence, detecting by adopting the high performance liquid chromatography condition determined in the step 1, determining by retention time, drawing a standard curve according to the corresponding relation between the peak area of each concentration and the concentration of the peak area, respectively determining the linear regression equation of the two flavanols,
the linear regression equation of the two flavanols is as follows:
catechin: 26.747x-9.338, R20.9917, x applies: 0.5-50 mug/mL;
epicatechin: 73.919x-37.145, R20.9928, x applies: 0.2-50 mug/mL;
wherein: y is the peak area corresponding to flavanols, x is the mass concentration of flavanols, R2Is a linear correlation coefficient;
step 6, detecting the actual vinegar sample
Diluting 5 actual vinegar samples by 10-20 times with distilled water, pretreating according to the method in the step 4, detecting by adopting the high performance liquid chromatography condition determined in the step 1, measuring peak areas of components in the samples, determining the qualitative by retention time, and calculating the contents of two flavanol substances, namely catechin and epicatechin in the actual vinegar samples according to the linear regression equation determined in the step 5.
2. The SPE-DES-HPLC method for simultaneously determining two flavanols in Shanxi mature vinegar as claimed in claim 1, wherein the method comprises the following steps: the eluent in the step 4 comprises absolute ethyl alcohol, 70% ethyl alcohol, ethyl acetate, methanol, DES with the molar ratio of tetraethyl ammonium chloride to n-octanoic acid being 1:2-51Tetrabutylammonium chloride and n-octylDES with acid molar ratio of 1:22And the molar ratio of choline chloride to acetic acid is 1:2 DES3Preferably DES with a 1:3 molar ratio of tetraethylammonium chloride to caprylic acid1。
3. The SPE-DES-HPLC method for simultaneously determining two flavanols in Shanxi mature vinegar as claimed in claim 1, wherein the method comprises the following steps: the optimal dosage of the XAD-2 macroporous adsorption resin activated in the step 4 is 188mg, and the optimal adsorption time is 11 min.
4. The SPE-DES-HPLC method for simultaneously determining two flavanols in Shanxi mature vinegar as claimed in claim 1, wherein the method comprises the following steps: the optimal dosage of the eluent in the step 4 is 400 mu L, and the optimal resolution time is 20 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110802713.XA CN113567578B (en) | 2021-07-15 | 2021-07-15 | SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110802713.XA CN113567578B (en) | 2021-07-15 | 2021-07-15 | SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113567578A true CN113567578A (en) | 2021-10-29 |
| CN113567578B CN113567578B (en) | 2022-09-23 |
Family
ID=78165013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110802713.XA Active CN113567578B (en) | 2021-07-15 | 2021-07-15 | SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113567578B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190336884A1 (en) * | 2016-12-29 | 2019-11-07 | Basf Beauty Care Solutions France Sas | Use of coconut water as extraction solvent |
| CN110824030A (en) * | 2019-08-27 | 2020-02-21 | 杭州师范大学 | Method for extracting pesticide from curcuma wenyujin |
| WO2020239929A1 (en) * | 2019-05-29 | 2020-12-03 | Assistance Publique - Hopitaux De Paris | Green tea catechins eutectic system |
| CN113063892A (en) * | 2021-03-17 | 2021-07-02 | 山东科技大学 | Method for extracting tea polyphenol by combining calculation and screening ternary natural eutectic solvent |
-
2021
- 2021-07-15 CN CN202110802713.XA patent/CN113567578B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190336884A1 (en) * | 2016-12-29 | 2019-11-07 | Basf Beauty Care Solutions France Sas | Use of coconut water as extraction solvent |
| WO2020239929A1 (en) * | 2019-05-29 | 2020-12-03 | Assistance Publique - Hopitaux De Paris | Green tea catechins eutectic system |
| CN110824030A (en) * | 2019-08-27 | 2020-02-21 | 杭州师范大学 | Method for extracting pesticide from curcuma wenyujin |
| CN113063892A (en) * | 2021-03-17 | 2021-07-02 | 山东科技大学 | Method for extracting tea polyphenol by combining calculation and screening ternary natural eutectic solvent |
Non-Patent Citations (7)
| Title |
|---|
| JI LI 等: "Efficient extraction of major catechins in Camellia sinensis leaves using green choline chloride-based deep eutectic solvents", 《RSC ADV.》》 * |
| NAJING FU 等: "Environmentally friendly and non-polluting solvent pretreatment of palm samples for polyphenol analysis using choline chloride deep eutectic solvents", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| ZHANG, JIN-HUA 等: "Studies on Adsorption Kinetics and Thermodynamics of Macroporous Resin for Rosmarinic Acid", 《JOURNAL OF OLEO SCIENCE》 * |
| 林慧 等: "高效液相色谱法测定树脂吸附对菠萝果汁中酚类物质的影响", 《食品研究与开发》 * |
| 阮怿航 等: "响应面法优化低共熔溶液提取儿茶素工艺及组分分析", 《食品工业》 * |
| 陈培云 等: "低共熔溶剂在食品样品前处理中的应用", 《食品安全质量检测学报》 * |
| 颜栋美 等: "高效液相色谱法测定金花茶中5种酚类物质的研究", 《河南工业大学学报(自然科学版)》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113567578B (en) | 2022-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2842957B1 (en) | Method for extracting and separating ginkgolides | |
| Zou et al. | Determination of indican, isatin, indirubin and indigotin in Isatis indigotica by liquid chromatography/electrospray ionization tandem mass spectrometry | |
| CN102818865A (en) | Method for Analyzing Oligomeric Proanthocyanidin (OPC) | |
| CN105181829B (en) | Rapid high-sensitivity synchronous quantitative determination method for leaf total folic acid and folic acid derivatives | |
| Jones et al. | Investigating sub-2 μm particle stationary phase supercritical fluid chromatography coupled to mass spectrometry for chemical profiling of chamomile extracts | |
| JP3386796B2 (en) | Quality determination method for plants of the genus Salicaceae and / or extracts thereof | |
| Zhao et al. | Quantitative analysis of betaine in Lycii Fructus by HILIC-ELSD | |
| CN109085285B (en) | Quality control method of changyanning granules | |
| CN104958330A (en) | Oroxylum indicum general flavone extraction and purification method and application thereof | |
| CN107315058A (en) | A kind of method of total ginkgoic acid in detection ginkgo biloba succi | |
| Wang et al. | An Efficient Strategy Based on Liquid–Liquid Extraction With Acid Condition and HSCCC for Rapid Enrichment and Preparative Separation of Three Caffeoylquinic Acid Isomers From Mulberry Leaves | |
| CN113567578B (en) | SPE-DES-HPLC method for simultaneously determining two flavanol substances in Shanxi mature vinegar | |
| CN111505162A (en) | Method for measuring content of 6 polyphenol compounds in compound red skin blood replenishing oral liquid | |
| Yang et al. | Simple and sensitive determination of sulfites in Chinese herbal teas by ultrahigh-performance liquid chromatography tandem mass spectrometry | |
| CN113533608B (en) | Method suitable for rapidly detecting aflatoxin in large-batch edible oil samples | |
| CN110687224B (en) | Method for measuring triptolide A in tripterygium wilfordii medicinal material and tripterygium wilfordii multi-glycoside tablet prepared from tripterygium wilfordii medicinal material | |
| CN108623642B (en) | Deep eutectic solvent water mixture for synchronously extracting salidroside and tyrosol from rhodiola rosea, and preparation method and extraction method thereof | |
| CN109917045B (en) | HPLC method for simultaneously measuring contents of 5 components in prepared rhizoma cibotii decoction pieces | |
| CN112666302A (en) | Method for identifying active flavone component group in barley seedling and rapidly detecting active flavone component group | |
| CN112438400A (en) | Method for purifying Meyer sedge total flavonoids and application thereof | |
| Cong et al. | Simultaneous determination of seven bioactive lignans in Herpetospermum caudigerum by RP‐HPLC method | |
| CN113899840B (en) | Method for establishing lignan component fingerprint of ginkgo leaf extraction intermediate or ginkgo leaf preparation and application thereof | |
| CN113156017B (en) | Method for simultaneously determining contents of 12 chemical components in strong dizzy-stop tablet by adopting HPLC (high performance liquid chromatography) | |
| CN113030329B (en) | One-test-multiple-evaluation method for determining content of 6 gingerol components in ginger juice brown sugar | |
| CN111830150B (en) | Method for determining content of flavonoid components in Ziziphora Bungeana Juz by one-test-multiple-evaluation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221215 Address after: 500m west of Dazhaozhuang Forest Farm, Shenchi County, Xinzhou City, Shanxi Province Patentee after: Shenchi haiziyuan vinegar Co.,Ltd. Address before: 030006 No. 92, Hollywood Road, Taiyuan, Shanxi Patentee before: SHANXI University |