CN109738562B - Method for synchronously detecting polyphenol in vegetable oil material by liquid chromatography-tandem mass spectrometry - Google Patents
Method for synchronously detecting polyphenol in vegetable oil material by liquid chromatography-tandem mass spectrometry Download PDFInfo
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Abstract
The invention provides a method for synchronously detecting polyphenol in vegetable oil by liquid chromatography-tandem mass spectrometry, which combines a magnetic solid phase extraction technology with a liquid chromatography-tandem mass spectrometry technology and is used for detecting phenolic compounds in vegetable oil, wherein the phenolic compounds are 2- (4-hydroxyphenyl) ethanol, cinnamic acid, p-coumaric acid, 2-hydroxycinnamic acid, vanillic acid, caffeic acid, ferulic acid, syringic acid, sinapyl alcohol, sinapic acid, trans-resveratrol, daidzein, apigenin, genistein, catechin, epicatechin, sesamin, daidzin and genistin. The invention is based on Fe3O4The RGO composite material can realize the detection and analysis of 19 phenolic compounds in the vegetable oil by utilizing a magnetic solid phase extraction method and combining liquid chromatography tandem mass spectrometry detection. The method is efficient, sensitive and stable, and is suitable for synchronous detection of phenolic compounds in the vegetable oil material.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a method for synchronously detecting polyphenol in vegetable oil materials by liquid chromatography-tandem mass spectrometry.
Background
Edible vegetable oil belongs to a fat-soluble medium, vegetable oil belongs to a solid-phase medium, forms of polyphenol compounds in different media have difference, extraction modes are different, and the method for synchronously detecting polyphenol in vegetable oil is mainly researched in this chapter. The vegetable oil is rich in nutrient substances such as phytosterol, tocopherol, flavonoid, phenols and the like, and the nutrient components are closely related to human health. The relevant documents report that phenolic compounds have good inoxidizability, anti-inflammation, antibacterial property, antianxiety property, anticancer property and the like, and people take proper phenolic substances every day to benefit the body. Therefore, it is of great interest to study these vegetable oils.
Most of the previous researches mainly adopt an ultraviolet-visible spectrophotometer method and a high performance liquid chromatography method to measure the content of total phenols in the vegetable oil materials, and therefore the oxidation resistance and the nutritional function are evaluated. With the development of analytical techniques, HPLC-ESI-MS2The plant oil nutrition powder is widely applied to the field due to good sensitivity, precision and accuracy, a large amount of monomer phenols are discovered one by one, such as the peanuts are rich in resveratrol, the soybeans are rich in isoflavones, the sesame is rich in sesamol, and the like, and a new thought is provided for developing the nutrition function of plant oil. However, the content of some phenolic compounds in the vegetable oil materials is extremely low, and the phenolic compounds in the samples need to be purified and enriched by adopting a proper method. The prior pretreatment method comprises solid-liquid extraction and ultrasonic assistanceAssisted extraction, microwave-assisted extraction, solid-phase extraction and the like, but the methods have the disadvantages of complicated operation, high cost, large pollution and the like. At present, a magnetic solid phase extraction method is developed and utilized gradually, and the method is simple, efficient, environment-friendly and economical and can be used for efficiently extracting phenolic compounds in complex samples.
Disclosure of Invention
The invention aims to provide a method for synchronously detecting polyphenol in vegetable oil materials by liquid chromatography-tandem mass spectrometry.
In order to achieve the purpose of the invention, the method for synchronously detecting the polyphenol in the vegetable oil material by the liquid chromatography-tandem mass spectrometry provided by the invention is used for detecting the phenolic compound in the vegetable oil material by combining a magnetic solid phase extraction technology and a liquid chromatography-tandem mass spectrometry technology, wherein the phenolic compound is at least one selected from 2- (4-hydroxyphenyl) ethanol, cinnamic acid, p-coumaric acid, 2-hydroxycinnamic acid, vanillic acid, caffeic acid, ferulic acid, syringic acid, sinapyl alcohol, sinapinic acid, trans-resveratrol, daidzein, apigenin, genistein, catechin, epicatechin, sesamin, daidzin and genistin. The method comprises the following steps:
1) preparation of a sample: crushing a sample to be detected to prepare a solution, and adding a ferroferric oxide-graphene material (Fe) into a reaction tube filled with the sample solution to be detected3O4-RGO) enrichment of the target, adsorbing solids with a magnet on the tube wall, discarding the liquid, eluting the residual solids with an analytical agent after removing impurities, centrifuging or filtering to collect the liquid, transferring the liquid to a centrifuge tube, drying with nitrogen and redissolving to complete the preparation of the sample;
2) preparation of standard solution: preparing standard substance solutions of various phenolic compounds with different concentrations;
3) detecting each standard solution by using a liquid chromatography tandem mass spectrometry, and respectively drawing a standard curve of each phenolic compound by using the concentration of the standard solution as an abscissa and using the signal intensity (total peak area) of the standard solution obtained by the mass spectrometry as an ordinate;
4) and (3) detecting the signal intensity of each phenolic compound in the sample solution sample to be detected according to the same conditions in the step 3), substituting the signal intensity into the standard curve to calculate to obtain the concentration of each target object in the sample solution to be detected, and realizing synchronous quantitative detection of the phenolic compounds contained in the vegetable oil material.
Preferably, the resolving agent in step 1) is methanol, preferably methanol containing 0.1% by volume fraction of formic acid.
Preferably, the nitrogen is dried and then redissolved by methanol in the step 1), and methanol is used for preparing each standard solution in the step 2).
In the foregoing method, step 1) specifically includes: weighing 1g of a sample to be detected in a centrifuge tube, adding 5mL of methanol aqueous solution with volume fraction of 70%, soaking the sample for 24h, then performing ultrasonic treatment at room temperature for 30min, and then centrifuging at 8000rpm for 10 min; drying 100 mu L of supernatant liquid with nitrogen, and re-dissolving with 2mL of methanol aqueous solution with volume fraction of 0.5%; then adding 4mg of ferroferric oxide-graphene material into the mixture, and carrying out vortex for 60 s; adsorbing the solid on the tube wall by using a magnet, and discarding the liquid; adding 1mL of petroleum ether into the residual solid, cleaning to remove impurities, adsorbing the solid on the tube wall by using a magnet, and discarding the liquid; adding 6mL of methanol containing formic acid with the volume fraction of 0.1% into the residual solid for elution, performing ultrasonic treatment at room temperature for 5min, centrifuging or filtering to collect liquid, transferring the liquid into a centrifuge tube, drying by nitrogen, redissolving by using 1mL of methanol, filtering by using a 0.22-micron organic membrane, and waiting for testing on a computer.
Preferably, the chromatographic conditions in step 3) are as follows: the liquid chromatographic column is Hypersil Gold C18Reverse phase chromatography column, 100mm × 2.1mm, 3.0 μm i.d.; mobile phase A: methanol containing 0.01% by volume of acetic acid, mobile phase B: acetic acid aqueous solution with volume fraction of 0.01%, gradient elution procedure as follows; the sample injection amount is 3 mu L, the flow rate is 200 mu L/min, and the column temperature is 30 ℃;
preferably, the mass spectrometric conditions in step 3) are as follows: adopting an electrospray ionization source ESI; SRM full scan mode; the scanning mode is a positive ion mode, the spraying voltage is 3500V, and the scanning time is 0.2 s; the scanning mode is negative ion mode, the spray voltage is-4000V, the scanning time is 0.05s, and the capillary temperature330 ℃ and the sheath gas is N235units, and the auxiliary gas is N25units, Ar as the collision gas, 1.0 mTorr.
According to the above chromatography, mass spectrometry (HPLC-ESI-MS)2) The conditions, retention time of each phenolic compound in the liquid chromatography in the step 3), and mass-to-charge ratio of parent ion and daughter ion of detection signal generated by each phenolic compound in the mass spectrum are respectively as follows:
the linear range, detection limit and quantification limit of the quantitative detection of each phenolic compound are respectively as follows:
in the present invention, the vegetable oil is selected from sesame, peanut, soybean, etc.
In the invention, the preparation method of the ferroferric oxide-graphene material comprises the following steps:
(1) accurately weighing 2.0g of graphite powder in 46mL of concentrated H2SO4(concentration 98%), stirring in ice-water bath for 30min, and adding 1.0g of NaNO3Mixing well, mixing 6.0g KMnO4Slowly adding into the mixture for 2h, heating to 35 deg.C, and reacting for 2 h. When the color of the solution changed from dark green to brown, 90mL of ultrapure water was added. The solution was then heated to 98 ℃ and 280mL of ultrapure water and 30% H were added2O210mL, and magnetic stirring for 60min to obtain the initial product. And (3) centrifugally separating the primary product at 8000rpm, washing the primary product for three times to be pure by using ultrapure water and a 5% hydrochloric acid solution respectively, and drying the washed product in vacuum to obtain Graphene Oxide (GO).
(2) 40mL of ethylene glycol was added to a dry beaker, 0.2g of Graphene Oxide (GO) prepared above was added, and the mixture was allowed to warm to room temperatureThe material was completely dissolved by ultrasonic agitation. 1.8g of sodium acetate (NaAc) and 0.7g of iron chloride (FeCl) were added3) And stirring for 30min to uniformly mix the reactants, transferring the reaction slurry into a 100mL polytetrafluoroethylene inner container, and pressurizing and sealing the reaction kettle. Placing the reaction kettle in a high-temperature box to react for 12h at 200 ℃, naturally cooling to room temperature after the reaction is finished, uncovering the reaction kettle, taking out the product, repeatedly washing the product for 5 times by using ultrapure water and absolute ethyl alcohol until the product is pure, freezing the product for 48h at-48 ℃ by vacuum freeze drying hydrazine to obtain a final product, and grinding the product into powder for later use.
Remarking: in the reaction process, keeping the reaction vessel dry, and completely mixing the materials; after the reaction, the product was transferred using a strong magnet.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention is based on Fe3O4The RGO composite material utilizes a magnetic solid phase extraction method and combines liquid chromatography tandem mass spectrometry detection to establish a set of simple, efficient, sensitive and accurate method for synchronously detecting 19 phenolic compounds in the vegetable oil material. The phenolic compounds in the vegetable oil are purified and enriched after the steps of ferroferric oxide-graphene composite material adsorption, impurity removal, elution and the like, and the phenol compounds are quantitatively analyzed by utilizing a liquid chromatography-tandem mass spectrum. With 4mg of Fe3O4RGO adsorbent, 2mL of 5% methanol-water dispersion solvent, 6mL of 0.1% formic acid-methanol elution solvent. According to methodology investigation, the method keeps the recovery rate of 19 phenolic compounds at 71.2% -116.6%, the detection limit is 0.02-90 mug/kg, the quantification limit is 0.06-300 mug/kg, the method has a good linear range, the day-to-day precision is lower than 10.8%, and the influence of the matrix is small. Therefore, the method is efficient, sensitive and stable, and is suitable for synchronous detection and analysis of phenolic compounds in the vegetable oil.
In actual sample detection, the average content of total phenols of the vegetable oil plants is 475.1-8155.2 mg/kg, sesame is rich in sesamin, peanuts are rich in 2- (4-hydroxyphenyl) ethanol, vanillic acid, catechin and epicatechin, and soybeans are rich in isoflavone compounds.
The experimental data show that the content of certain phenolic compounds in the same type of vegetable oil is influenced by varieties, and the black oilseeds have higher nutritive value, so that the invention provides a new idea for the subsequent development of the black oilseed industry.
Drawings
FIG. 1 is a diagram of the extraction of phenolic compounds from vegetable oil in accordance with the present invention as described in example 2.
FIG. 2 is a graph showing the effect of the amount of adsorbent used on phenolic compound extraction in example 2 of the present invention.
FIG. 3 shows the effect of the type of dispersing solvent on the extraction of phenolic compounds in example 2 of the present invention.
FIG. 4 is a graph showing the effect of the amount of dispersing solvent on the extraction of phenolic compounds in example 2 of the present invention.
FIG. 5 shows the effect of elution solvent on phenolic compound extraction in example 2 of the present invention.
FIG. 6 shows the effect of elution solvent dosage on phenolic compound extraction in example 2 of the present invention.
FIG. 7 shows the effect of the addition of formic acid as the elution solvent on the extraction of phenolic compounds in example 2 of the present invention.
FIG. 8 is a chromatogram of a mixed standard solution of 1mg/kg of phenolic compounds in example 2 of the present invention.
FIG. 9 is a graph showing the detection limit of the mixed standard solution of phenolic compounds in example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The main reagents used in the present invention are shown in table 1.
TABLE 1 Main reagents
The instruments and consumables used are shown in Table 2.
TABLE 2 Main instruments and consumables
Example 1 ferroferric oxide-graphene (Fe)3O4Preparation of-RGO) adsorbent Material
1. Accurately weighing 2.0g of graphite powder in 46mL of concentrated H2SO4(concentration 98%), stirring in ice-water bath for 30min, and adding 1.0g of NaNO3Mixing well, mixing 6.0g KMnO4Slowly adding into the mixture for 2h, heating to 35 deg.C, and reacting for 2 h. When the color of the solution changed from dark green to brown, 90mL of ultrapure water was added. The solution was then heated to 98 ℃ and 280mL of ultrapure water and 30% H were added2O210mL, and magnetic stirring for 60min to obtain the initial product. And (3) centrifugally separating the primary product at 8000rpm, washing the primary product for three times to be pure by using ultrapure water and a 5% hydrochloric acid solution respectively, and drying the washed product in vacuum to obtain Graphene Oxide (GO).
2. 40mL of ethylene glycol was added to a dry beaker, 0.2g of Graphene Oxide (GO) prepared above was added and the material was completely dissolved by ultrasonic stirring at room temperature. 1.8g of sodium acetate (NaAc) and 0.7g of iron chloride (FeCl) were added3) And stirring for 30min to uniformly mix the reactants, transferring the reaction slurry into a 100mL polytetrafluoroethylene inner container, and pressurizing and sealing the reaction kettle. Placing the reaction kettle in a high-temperature box to react for 12h at 200 ℃, naturally cooling to room temperature after the reaction is finished, uncovering the reaction kettle, taking out the product, repeatedly washing the product for 5 times by using ultrapure water and absolute ethyl alcohol until the product is pure, freezing the product for 48h at-48 ℃ by vacuum freeze drying hydrazine to obtain a final product, and grinding the product into powder for later use.
Remarking: in the reaction process, keeping the reaction vessel dry, and completely mixing the materials; after the reaction, the product was transferred using a strong magnet.
Example 2 method for synchronously detecting polyphenol in vegetable oil material by liquid chromatography-tandem mass spectrometry
(I) preparation of mixed standard solution and quality control sample
1. Mixed Standard solution preparation
Accurately weighing 19 standard substances of 10.00-15.00 (+/-0.01) mg of 2- (4-hydroxyphenyl) ethanol, cinnamic acid, p-coumaric acid, 2-hydroxycinnamic acid, vanillic acid, caffeic acid, ferulic acid, syringic acid, sinapyl alcohol, sinapic acid, trans-resveratrol, daidzein, apigenin, genistein, catechin, epicatechin, sesamin, daidzin and genistin in a volumetric flask, accurately transferring a certain amount of methanol solution into the volumetric flask by using a liquid transfer gun, completely dissolving and uniformly mixing the standard substances after ultrasonic vortex, preparing a single standard solution of 1.00mg/mL, sealing, and storing in a refrigerator at-20 ℃ for later use.
Respectively transferring 0.1mL of the 19 single standard solutions into a 20mL volumetric flask, then adding 8.1mL of methanol, uniformly mixing to obtain a 10 mu g/mL mixed standard solution, diluting with methanol step by step according to the method to prepare mixed standard solutions with different concentration series, and storing in a refrigerator at-20 ℃ for later use.
2. Quality control sample preparation
(1) Collecting samples:
in order to avoid the sample singularity, 76 vegetable oil samples such as black sesame, white sesame, black peanut, safflower, black bean and soybean are collected from Jilin, Anhui, Henan, Hubei and Shandong provinces in China (Table 3).
TABLE 3 oilseed sample Collection information
(2) Sample preparation: drying the collected sample, mashing the sample into powder by a slicer and a pulverizer, storing by a sealed bag, and sticking a label for later use.
(3) Preparing a quality control sample: selecting one of semen Sesami Niger, semen Sesami Indici, semen Arachidis Hypogaeae, Carthami flos, semen Sojae Atricolor and semen glycines, weighing 10.00 (+ -0.01) g of each sample in a sealed bag, shaking repeatedly, and mixing.
(II) HPLC-ESI-MS2Method establishment
(1) Chromatographic conditions
The chromatographic column is Hypersil Gold C18Reverse phase chromatography column (100mm × 2.1mm, 3.0 μm i.d.); mobile phase A: 0.01% acetic acid-methanol solution (v: v), mobile phase B: 0.01% acetic acid-water solution (v: v), gradient elution procedure is shown in Table 4. The sample volume was 3. mu.L, the flow rate was 200. mu.L/min, and the column temperature was 30 ℃.
TABLE 4 gradient elution procedure
(2) Conditions of Mass Spectrometry
Using an electrospray ion source (ESI); SRM full scan mode; in a scanning mode positive ion mode (positive), the spray voltage is 3500V, and the scanning time is 0.2 s; scanning negative ion mode (negative), spray voltage is-4000V, scanning time is 0.05s, capillary temperature is 330 deg.C, and sheath gas (N)2)35units, supplement gas (N)2)5units, 1.0mTorr of impinging gas (Ar).
The response degree of the analyte in the mass spectrometer can be improved by selecting a proper primary-secondary ion pair, collision energy, lens voltage and the like, so that the sensitivity is improved. Therefore, 19 phenol standards with the concentration of 1.00 mu g/mL are directly injected by mass spectrometry by using a flow injection method, and positive ions [ M + H ] are respectively selected]+Mode and anion [ M-H ]]-Mode (table 5).
TABLE 5 analyte HPLC-ESI-MS2Parameter(s)
(III) Standard Curve construction
Respectively transferring 1mL of mixed standard solution with different concentrations into a sample injection bottle, and selecting the established HPLC-ESI-MS2The method is used for performing on-machine test, the concentration of each standard solution is taken as a horizontal coordinate, the corresponding peak area is taken as a vertical coordinate, and a standard working curve is drawn after linear regression.
(IV) establishment of pretreatment method
Accurately weighing 1.00 (+ -0.01) g of quality control sample in a centrifuge tube, adding 5mL of 70% methanol-water solution, soaking the sample for 24h, then performing ultrasonic treatment at normal temperature for 30min, and centrifuging the sample at 8000rpm for 10 min. After 100. mu.L of the supernatant was dried with nitrogen, it was redissolved in 2mL of 0.5% methanol-water solution. Then adding 4mg of ferroferric oxide-graphene adsorbent into the redissolution, and carrying out vortex for 60 s; the strong magnet is externally used on the tube wall to attract the material, and the solution is discarded; removing impurities from the material by using 1mL of petroleum ether, attracting the material outside the tube wall by using a magnet, and abandoning the solution; adding 6mL of methanol solution to elute the material, performing ultrasonic treatment at room temperature for 5min, retaining the solution, transferring the solution into a 10mL centrifuge tube, drying by nitrogen, redissolving by 1mL of methanol solution, filtering by a 0.22 mu m organic membrane, and loading on the machine. The pretreatment process is shown in figure 1.
1. Selection of the quantity of adsorbent
Ferroferric oxide-graphene (Fe)3O4the-RGO) composite material has good specific surface area, and the surface of the material has carboxyl, hydroxyl and the like, and can form pi-pi bond intermolecular force with phenolic compounds to attract each other. When the adsorbent is insufficient, the target cannot be completely adsorbed; on the contrary, when the adsorbent is excessive, the target substance is hardly eluted. Therefore, the dosage of the adsorbent has great influence on the extraction of the phenolic compounds in the sample, and the invention adopts 2, 4, 6, 8 and 10mg of adsorbing materials for research respectively. The results of the experiment are shown in FIG. 2, where the total peak area is maximal when the material is used in an amount of 4 mg. Thus, the ferroferric oxide-graphene (Fe) of the present invention3O4The optimum amount of-RGO) adsorbent material was 4 mg.
2. Selection of concentration of dispersing solvent
Methanol-water is adopted as a dispersing solvent, wherein the ratio of methanol to water is proper, and two conditions are met: firstly, the solvent can completely dissolve the phenolic compound; and secondly, the intermolecular acting force between the phenolic compound and the solvent is small, so that the ferroferric oxide-graphene composite material can be adsorbed with a target better. The invention carries out comparative study by using five methanol-water (v: v) dispersion solvents with different proportions of 0 percent, 2.5 percent, 5 percent, 10 percent and 20 percent respectively. As shown in FIG. 3, the total peak area is the largest when 5% methanol-water (v: v) is used. Therefore, 5% methanol-water (v: v) was used as the dispersion solvent of the present invention.
3. Selection of the amount of dispersing solvent
The proper amount of the dispersing solvent is selected to ensure that the material is uniformly dispersed in the solvent and the adsorption effect is ensured. However, when the amount of the dispersion solvent is excessive, the interaction between the material and the target molecule is not strong, and the target cannot be completely adsorbed. The conditions of the experiment are optimized by selecting 2, 3, 4, 5 and 6mL of dispersing solvent. As is apparent from fig. 4, when the amount of the dispersion solvent was 2mL, the total peak area was the largest, and when the amount exceeded 3mL, the material adsorption efficiency was significantly decreased. Therefore, in the present invention, the amount of the dispersion solvent is 2mL in consideration of the adsorption efficiency and the solvent consumption.
4. Selection of elution solvent species
Four elution solvents, acetone, methanol, ethanol and acetonitrile were investigated. As shown in fig. 5, when the elution solvent was methanol, the total peak area was the highest. Therefore, methanol is used as the elution solvent in the present invention.
5. Choice of elution solvent dosage
The material adsorbs the target through intermolecular force, the target can be completely eluted by using a proper amount of the eluent, the waste of the solvent can be avoided, in order to research the influence of the amount of the eluting solvent on the desorption efficiency, the invention adopts 1mL, 2mL, 3mL, 4 mL, 6mL and 8mL of the eluting solvent to carry out experiments, the result is shown in FIG. 6, and when the volume of the eluent is 6mL, the total peak area is the highest. Therefore, the dosage of the elution solvent used in the invention is 6 mL.
6. Selecting the amount of formic acid added in the elution solvent
Considering that the pi-pi intermolecular force exists between the material and the phenolic compound, belonging to chemical adsorption, a certain amount of formic acid is added into the eluent to improve the desorption efficiency. Thus, the present invention employs five different ratios of formic acid-methanol (v: v) eluents of 0%, 0.05%, 0.1%, 0.2%, and 0.5%. As is evident from FIG. 7, the total peak area is maximal when 0.1% formic acid-methanol (v: v) eluent is selected. Therefore, the final elution solvent of the present invention was 0.1% formic acid-methanol (v: v).
Through the analysis, the pretreatment method adopted by the invention comprises the following steps: 4mg of ferroferric oxide-graphene adsorption material, 2mL of 5% methanol-water (v: v) dispersion solvent, and 6mL of 0.1% formic acid-methanol (v: v) elution solvent (resolving agent).
(V) methodological verification
1. Linear range and sensitivity
By using optimized HPLC-ESI-MS2The test method is shown in the (II) HPLC-ESI-MS2And (5) establishing a method. Quantitative analysis is carried out on the standard solution of the phenolic compound with different concentrations, and the detection Limit (LOD), the quantitative Limit (LOQ), the linear range and the like of the method are considered. Taking the concentration (X) of the standard solution of the analyte as a horizontal coordinate and the peak area (Y) as a coordinate, and performing one-dimensional linear equation regression analysis by adopting a 6-9 point method; calculating the detection limit and the quantification limit of the target object by using a 3-time signal-to-noise ratio (S/N is 3) and a 10-time signal-to-noise ratio (S/N is 10); with R2Above 0.98 a linear range was established and the results are shown in table 6. The method has detection limit of 0.02-90 μ g/kg, quantitative limit of 0.06-300 μ g/kg, linear range of 1-20000 μ g/kg, and linear correlation coefficient R2Are all above 0.9861. FIG. 8 is a spectrum of a mixed standard solution of 1mg/kg, the peaks of 19 phenolic compounds are sharp, the separation condition is good, and the analysis time only needs 16 min; FIG. 9 is a spectrum of detection limits of individual phenolic compounds, with distinct peaks within corresponding retention times, enabling baseline separation.
TABLE 6 working curves, linear ranges, detection limits, quantitation limits for phenolic compounds
2. Precision and accuracy
In order to examine the precision and accuracy of the method, three levels of standard substances of 50 mug/kg, 200 mug/kg and 500 mug/kg are added into a quality control sample, and the intra-day precision, the inter-day precision and the adding standard recovery rate are measured. Day precision is the Relative Standard Deviation (RSD) of five consecutive replicates of a sample; day precision is the Relative Standard Deviation (RSD) of the results of five consecutive days of the sample; the recovery rate of the spiked sample is the average of three consecutive measurements of the spiked sample, and the results are shown in Table 7. The recovery rate of the standard addition with different concentrations is kept between 71.2 and 116.6 percent, and the precision in and between days is less than 10.8 percent. The method has good precision and accuracy, and can be used for accurately and quantitatively analyzing the phenolic compounds in the sample.
Precision and accuracy of the methods of Table 7
Note: ND represents no detection, and is lower than the detection limit.
3. Matrix effect
In chemical analysis, a matrix refers to a component other than a target in a sample. The matrix often interferes significantly with the analysis of the target and affects the accuracy of the analysis results. Therefore, the invention adopts a standard addition method to examine the influence of the matrix effect. To the blank substrate sample (A), 200. mu.g/kg of the mixed standard solution (B) was added to prepare a solution C. And comparing the content relationship among the matrix solution (A), the standard solution (B) and the matrix solution (C) added with the standard solution to obtain a matrix effect.
Matrix Effect (ME) calculation formula:
the results are shown in Table 8, with the matrix effect remaining at-13% to 8%. The method shows that after the pretreatment method is used for purification and enrichment, the influence of the matrix in the sample on the analysis of the target object is small, and the quantitative analysis of the phenolic compounds in the actual sample can be met.
TABLE 8 matrix Effect of phenolic Compounds in oilseed samples
4. Detection method comparison
The assay technique was compared with literature-related quantitative methods for the detection of oilseed phenolic compounds, and the results are shown in Table 9. The method can be used for synchronously analyzing 19 phenolic compounds, the analysis time is only 16min, the quantification limit is as low as 0.1 mu g/kg, and compared with other detection methods, the detection method has the advantages of short time consumption, high sensitivity and the like.
TABLE 9 comparison of detection methods for phenolic compounds in oilseeds
EXAMPLE 3 actual sample determination
The analysis method optimized in example 2 was adopted to quantitatively analyze 19 phenolic compounds in 76 vegetable oil samples, and the content distribution of the phenolic compounds is shown in table 10. Taking 10mg/kg as a nutrition evaluation standard, wherein sesameseed is mainly sesamin; the peanut mainly comprises 2- (4-hydroxyphenyl) ethanol, vanillic acid, catechin and epicatechin; the soybean mainly comprises isoflavone compounds such as daidzein, genistein, daidzin, genistin, etc. Wherein, the sesamin content in the black sesame is obviously higher than that of the white sesame; the content of trans-resveratrol in the black peanuts is obviously higher than that of the safflower; the isoflavone content of the black beans is obviously higher than that of the soybeans. Therefore, the content of certain phenolic compounds in the vegetable oil is influenced by the variety, and the black oilseeds have higher nutritional value from special nutritional ingredients, so that a thought is provided for the subsequent development of the black oilseed industry.
TABLE 10 phenolic Compound content in oilseeds
Note: ND represents no detection, and is lower than the detection limit.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Reference to the literature
1、Alu’Datt M H,Rababah T,Alhamad M N,et al.Characterization and antioxidant activities of phenolic interactions identified in byproducts of soybean and flaxseed protein isolation[J].Food Hydrocolloids,2016,61:119-127.
2、Dubie J,Stancik A,Morra M,et al.Antioxidant Extraction from Mustard(Brassica juncea)Seed Meal Using High-Intensity Ultrasound[M].2013.
3、
A,
-Czerniak A.Development of a novel gold nano particle-based method to determine antioxidant capacity of Brassica oilseeds,white flakes and meal[J].Food Chemistry,2016,208:142-149.
4、Sumczynski D,KotáskováE,OrsavováJ,et al.Contribution of individual phenolics to antioxidant activity and in vitro digestibility of wild rices(Zizania aquatica L.)[J].Food Chemistry,2017,218:107-115.
5、Siger A,Nogala-
M,Lampart-Szczapa E,et al.
fenolowych w nowych odmianach rzepaku[J]. 
fenolowych w nowych odmianach rzepaku-ResearchGate,2004,25(1):251-262。
Claims (5)
1. The method for synchronously detecting the polyphenol in the vegetable oil material by the liquid chromatography-tandem mass spectrometry is characterized in that the method combines a magnetic solid phase extraction technology with the liquid chromatography-tandem mass spectrometry technology and is used for detecting phenolic compounds in the vegetable oil material, wherein the phenolic compounds are 2- (4-hydroxyphenyl) ethanol, cinnamic acid, p-coumaric acid, 2-hydroxycinnamic acid, vanillic acid, caffeic acid, ferulic acid, syringic acid, sinapyl alcohol, sinapic acid, trans-resveratrol, daidzein, apigenin, genistein, catechin, epicatechin, sesamin, daidzin and genistin; the method comprises the following steps:
1) preparation of a sample: crushing a sample to be detected to prepare a solution, adding a ferroferric oxide-graphene material into a reaction tube filled with the solution of the sample to be detected to enrich a target object, adsorbing a solid on the tube wall by using a magnet, discarding the liquid, removing impurities from the residual solid, eluting the residual solid by using an analytic agent, centrifuging or filtering to collect the liquid, transferring the liquid into a centrifugal tube, drying by using nitrogen, and redissolving to finish the preparation of the sample;
2) preparation of standard solution: preparing standard substance solutions of various phenolic compounds with different concentrations;
3) detecting each standard solution by using a liquid chromatography tandem mass spectrometry, and respectively drawing a standard curve of each phenolic compound by using the concentration of the standard solution as an abscissa and using the signal intensity of the standard solution obtained by the mass spectrometry as an ordinate;
4) detecting the signal intensity of each phenolic compound in the sample solution sample to be detected according to the same conditions in the step 3), substituting the signal intensity into the standard curve for calculation to obtain the concentration of each target object in the sample solution to be detected, and realizing synchronous quantitative detection of the phenolic compounds contained in the vegetable oil material;
the step 1) is specifically as follows: weighing 1g of a sample to be detected in a centrifuge tube, adding 5mL of methanol aqueous solution with volume fraction of 70%, soaking the sample for 24h, then performing ultrasonic treatment at room temperature for 30min, and then centrifuging at 8000rpm for 10 min; drying 100 mu L of supernatant liquid with nitrogen, and re-dissolving with 2mL of methanol aqueous solution with volume fraction of 0.5%; then adding 4mg of ferroferric oxide-graphene material into the mixture, and carrying out vortex for 60 s; adsorbing the solid on the tube wall by using a magnet, and discarding the liquid; adding 1mL of petroleum ether into the residual solid, cleaning to remove impurities, adsorbing the solid on the tube wall by using a magnet, and discarding the liquid; adding 6mL of methanol containing formic acid with the volume fraction of 0.1% into the residual solid for elution, performing ultrasonic treatment for 5min at room temperature, centrifuging or filtering to collect liquid, transferring the liquid into a centrifuge tube, drying by nitrogen, redissolving by using 1mL of methanol, filtering by using a 0.22-micron organic membrane, and waiting for testing on a computer;
step 1) the resolving agent is methanol containing 0.1% by volume of formic acid;
the vegetable oil is selected from sesame, peanut and soybean;
the preparation method of the ferroferric oxide-graphene material comprises the following steps:
(1) 2.0g of graphite powder was weighed into 46mL of concentrated H2SO4In the process, after stirring in ice-water bath for 30min, 1.0g of NaNO is added3Mixing well, mixing 6.0g KMnO4Slowly adding into the mixture, maintaining for 2h, heating to 35 deg.C, and reacting for 2 h; when the color of the solution changes from dark green to brown, 90mL of ultrapure water is added; the solution was then heated to 98 ℃ and 280mL of ultrapure water and 30% H were added2O210mL, magnetically stirring for 60min to obtain a primary product; centrifuging the primary product at 8000rpm, washing and precipitating with ultrapure water and 5% hydrochloric acid solution for 3 times, and vacuum drying to obtain graphene oxide;
(2) adding 40mL of ethylene glycol into a dry beaker, adding 0.2g of the prepared graphene oxide, and performing ultrasonic stirring at room temperature to completely dissolve the material; adding 1.8g of sodium acetate and 0.7g of ferric chloride, stirring for 30min, uniformly mixing reactants, transferring the reaction slurry into a 100mL polytetrafluoroethylene inner container, and pressurizing and sealing the reaction kettle; placing the reaction kettle in a high-temperature box to react for 12h at 200 ℃, naturally cooling to room temperature after the reaction is finished, uncovering the reaction kettle, taking out the product, repeatedly washing the product for 5 times by using ultrapure water and absolute ethyl alcohol, freezing the hydrazine for 48h at-48 ℃ by vacuum freeze drying to obtain a final product, and grinding the final product into powder for later use;
the chromatographic conditions in step 3) are as follows: the liquid chromatographic column is Hypersil Gold C18Reverse phase chromatography column, 100mm × 2.1mm, 3.0 μm i.d.; mobile phase A: methanol containing 0.01% by volume of acetic acid, mobile phase B: acetic acid aqueous solution with volume fraction of 0.01%, gradient elution procedure as follows; the sample injection amount is 3 mu L, the flow rate is 200 mu L/min, and the column temperature is 30 ℃;
2. the method of claim 1, wherein the nitrogen is dried in step 1) and then reconstituted with methanol, and the solutions of each standard are prepared with methanol in step 2).
3. The method according to claim 1, wherein the mass spectrometric conditions in step 3) are as follows: adopting an electrospray ionization source ESI; SRM full scan mode; the scanning mode is a positive ion mode, the spraying voltage is 3500V, and the scanning time is 0.2 s; the scanning mode is negative ion mode, the spray voltage is-4000V, the scanning time is 0.05s, the capillary temperature is 330 ℃, and the sheath gas is N235units, and the auxiliary gas is N25units, Ar as the collision gas, 1.0 mTorr.
4. The method of claim 1, wherein the retention time of each phenolic compound in the liquid chromatography in step 3) and the mass-to-charge ratio of the parent ion and the daughter ion of the detection signal generated by each phenolic compound in the mass spectrum are respectively as follows:
5. the method of claim 1, wherein the linear range, detection limit and quantitation limit of the quantitative detection of each phenolic compound are as follows:
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323557A (en) * | 2013-06-16 | 2013-09-25 | 中国科学院成都生物研究所 | Method for analyzing isoflavone in soybeans and products thereof |
| CN104330498A (en) * | 2014-11-20 | 2015-02-04 | 中国农业科学院油料作物研究所 | Method for measuring polyphenol monomers in rapeseeds and processed products of rapeseeds by use of ultra-high performance liquid chromatography |
| CN108645815A (en) * | 2018-08-13 | 2018-10-12 | 中国农业科学院油料作物研究所 | A kind of near infrared detection method of content of fatty acid in vegetable oil material |
| CN108854154A (en) * | 2018-06-27 | 2018-11-23 | 浙江大学 | A kind of peanut shell polyphenol selective enrichment method using magnetic microsphere |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014186874A1 (en) * | 2013-05-23 | 2014-11-27 | Yyz Pharmatech, Inc. | Methods and compositions for enzyme linked immuno and hybridization mass spectrometric assay |
| DE102013218818A1 (en) * | 2013-09-19 | 2015-03-19 | Agilent Technologies, Inc. - A Delaware Corporation - | HPLC pump with active mixing element |
-
2018
- 2018-12-14 CN CN201811532659.6A patent/CN109738562B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323557A (en) * | 2013-06-16 | 2013-09-25 | 中国科学院成都生物研究所 | Method for analyzing isoflavone in soybeans and products thereof |
| CN104330498A (en) * | 2014-11-20 | 2015-02-04 | 中国农业科学院油料作物研究所 | Method for measuring polyphenol monomers in rapeseeds and processed products of rapeseeds by use of ultra-high performance liquid chromatography |
| CN108854154A (en) * | 2018-06-27 | 2018-11-23 | 浙江大学 | A kind of peanut shell polyphenol selective enrichment method using magnetic microsphere |
| CN108645815A (en) * | 2018-08-13 | 2018-10-12 | 中国农业科学院油料作物研究所 | A kind of near infrared detection method of content of fatty acid in vegetable oil material |
Non-Patent Citations (6)
| Title |
|---|
| Analysis of flavors and fragrances by HPLC with Fe3O4@GO magnetic nanocomposite as the adsorbent;Rui Xiao 等;《Talanta》;20170125;第166卷;第262-267页 * |
| Magnetic three-dimensional graphene nanoparticles for the preconcentration of endocrine-disrupting phenols;Li Liu 等;《Microchim Acta》;20140329;第1-7页 * |
| Rapid and effective sample cleanup based on graphene oxide-encapsulated core-shell magnetic microspheres for determination of fifteen trace environmental phenols in seafood by liquid chromatographye tandem mass spectrometry;Sheng-Dong Pan 等;《Analytica Chimica Acta》;20161231;第919卷;第34-46页 * |
| Simultaneous determination of phenolic compounds in sesame oil using LC–MS/MS combined with magnetic carboxylated multi-walled carbon nanotubes;Rao Wu 等;《Food Chemistry》;20160215;第204卷;第334-342页 * |
| 液相色谱- 质谱联用法分析葡萄籽提取物中的5种多酚类成分;杨代晓 等;《药物分析杂志》;20161231;第36卷(第8期);第1330-1337页 * |
| 石墨烯量子点磁性复合纳米粒子分散固相微萃取-毛细管电泳法测定肉桂酸及其衍生物;孙亚明 等;《色谱》;20170331;第35卷(第3期);第325-331页 * |
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