CN110220997B - Method for detecting antioxidant in fish meat - Google Patents
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Abstract
The invention discloses a method for detecting antioxidants in fish meat, relates to a method for detecting pollutants in food, is developed aiming at the detection method of aniline antioxidants in fish meat, and detects the concentration of the aniline antioxidants in the fish meat through the processes of extraction and purification instrument analysis and the like, and the method provided by the invention is realized through the following means: making an antioxidant standard curve; preparing a sample solution to be detected: pretreating, extracting, purifying and further purifying a fish sample to be detected to obtain a sample liquid to be detected; and (3) detecting and analyzing the sample liquid to be detected by using a gas chromatography-tandem triple quadrupole mass spectrometry (GC-EI-MS/MS), and calculating to obtain the content percentage of each component in the fish sample to be detected. The method has low detection limit and high recovery rate, and can be used for qualitative and quantitative detection of antioxidants in actual fish.
Description
Technical Field
The invention relates to a method for detecting pollutants in food, in particular to a method for detecting antioxidants in fish.
Background
Antioxidants (Antioxidants) are substances that block, inhibit or retard the oxidation or autoxidation of polymers. It can trap and neutralize various free radicals, such as O 2- OH and HO 2 And the like, thereby reducing or eliminating damage to the polymer and the human body. The antioxidant can obviously improve the oxidation stability of some oil products such as gasoline, diesel oil and the like and prolong the service time of the polymer. In the end of the 19 th century to the beginning of the 20 th century, antioxidants are widely used in related industries for improving the quality of oil products, rubber, plastics and other fields. Such as to avoid the formation of rubber vulcanization, metal corrosion, build-up of fouling of internal combustion engines formed by polymerization of fuel, and the like. In the 70 th century of 19 th century, bogge adopts p-phenylenediamine and beta-aniline in amine substances as antioxidant degradation agents of rubber products, so that the service time of rubber is greatly prolonged, but the antioxidant concept is not proposed by Moureon for the first time until the 20 th century. In the subsequent development of more than 100 years, new antioxidant varieties are continuously appeared, and the development of modern industry, such as the development of novel materials, automobiles and the like, is promoted.
In 1937, the antioxidant 2, 6-di-tert-butyl-4-methylphenol (BHT) which is the first hindered phenol structure in the world was discovered, and BHT was also developed in China in the early 80's of the last century. The investigation result at the end of the 80 s in the last century shows that the total consumption of plastic antioxidants in China is less than 1 kiloton, which is much lower than that of some western countries. By 2002, the total consumption of plastic antioxidants in china exceeded 2 million tons, approaching the united states, western europe, and other countries in the late 80 s of the 20 th century. At present, the variety and quality of the antioxidant which is independently researched and developed in China can basically meet the requirements of plastics and fuel industries in China, and on the basis, china exports some antioxidant products every year. The three most common antioxidants on the market are mainly phenolic antioxidants, phosphorus antioxidants and aminic antioxidants.
With the use of the antioxidants becoming more and more extensive, the environmental pollution characteristics of the antioxidants are changing constantly, and part of the antioxidants have difficult degradability and biological enrichment, such as aniline antioxidants. These target pollutants enter the natural water body and start to gradually accumulate, so that the original concentration of the trace pollutants is increased by several times, and the microorganisms, animals and plants in the natural water body generate resistance. In nature, some antioxidants are difficult to degrade, and it is reported that the antioxidants eventually enter the human body along with the food chain, so that the human body is carcinogenic and has an increased frequency of endocrine disorders. With the development of new drugs and the improvement of living standard of people, more antioxidants are generated in more economically developed cities. However, there are few studies on the detection of antioxidants such as aniline and its derivatives, and no information and database can be used to look up the content of these substances in the existing environmental water, so the detection method of antioxidants and the content in the environment become important research targets. With the improvement of the scientific and technological level, more and more chemicals are invented and applied, but the toxicological effects are not clear and are not strictly controlled, so that the chemicals enter the natural environment including the atmosphere, the soil, the surface water system in a large amount to further affect wild organisms (fish, shrimps, birds and the like). At present, no effective detection method aiming at the antioxidant ingredients in fish meat is reported, and no detection method for simultaneously detecting a plurality of antioxidant ingredients in fish meat is available.
Disclosure of Invention
The invention develops a detection method for aniline antioxidants in fish meat, and detects the concentration of the aniline antioxidants in the fish meat through processes such as extraction and purification instrument analysis and the like.
The method for detecting the antioxidant in the fish meat provided by the invention comprises the following steps:
(1) Making a standard curve: respectively preparing a gradient concentration standard solution of each antioxidant standard substance, respectively using gas chromatography-tandem triple quadrupole mass spectrometry (GC-EI-MS/MS) detection analysis, calculating the area of each component, and performing linear regression on the actual concentration of each component in the standard solution by using the peak area of each component to obtain a standard curve;
(2) Preparing a sample solution to be detected: pretreating, extracting, purifying and further purifying a fish meat sample to be detected to obtain a sample liquid to be detected;
(3) And (3) detecting and analyzing the sample liquid to be detected obtained in the step (2) by using gas chromatography-tandem triple quadrupole mass spectrometry (GC-EI-MS/MS), calculating to obtain peak areas of all components, substituting the peak areas of all the components into respective standard curve equations to obtain the actual concentration of all the components in the sample liquid to be detected, and further calculating to obtain the actual content of all the components in the fish sample to be detected and the content percentage of all the components in the fish sample to be detected.
The above steps are performed in the order of steps (1), (2) and (3), or in the order of steps (2), (1) and (3).
Preferably, the step (2) further comprises the following specific steps:
1) Fish meat pretreatment: taking a fish sample to be detected, adding anhydrous sodium sulfate, mixing, grinding into fine powder, adding an internal standard, and mixing uniformly;
2) And (3) extraction: adding a solvent into the pretreated fish sample for extraction, and concentrating the extract into a crude sample solution;
3) Purifying: purifying the crude sample solution by a gel chromatographic column, performing rotary evaporation, concentrating by a nitrogen blowing instrument, and adding isooctane to obtain a primary purified sample solution;
4) Further purification: and (3) loading the primary purified sample liquid to a purification column filled with silica gel and anhydrous sodium sulfate, eluting, adding a solvent, and concentrating to obtain the sample liquid to be detected.
Step 1), the mass ratio of the fish sample to be detected to the anhydrous sodium sulfate is 1; the internal standard is 13 C 12 DPA, the mass ratio of the added mass of the internal standard to the fish sample to be detected is 0.1 multiplied by 10 -6 5, preparing a mixture; and finally, uniformly mixing, standing and balancing for 20min.
The step 2) of extraction specifically comprises the following steps: adding solvent, shaking, collecting extractive solution, repeating above steps for 2 times, mixing extractive solutions, adding isooctane, and concentrating.
The solvent is an acetone/n-hexane mixed solvent, and the volume ratio of acetone/n-hexane in the mixed solvent is 1; the ratio of the volume of the solvent added in each extraction to the mass of the fish sample to be detected is 5mL; the oscillation time is 30min each time; after shaking extraction, centrifuging for 15 minutes by using a centrifugal machine at the rotating speed of 3000r/min, collecting the upper layer, then repeating the extraction step for 2 times, and combining the upper layers collected by three times of extraction to obtain extract liquor; the volume of the added isooctane is 1/5 of the volume of the solvent added each time in the extraction process; and (4) concentrating, wherein the volume after concentration is 1-2mL.
The purification of the gel chromatographic column in the step 3) specifically comprises the following steps: the crude sample solution was applied to a gel column (GPC, 30 mm. Times.10mm, biobead S-X3), and the objective contaminant was eluted with a mixture of ethyl acetate and cyclohexane in a volume ratio of 1. The volume of the added isooctane is 1-2mL.
Step 4), filling a column material of the purification column, wherein the column material is 5% of inactivated silica gel and anhydrous sodium sulfate, the mass ratio of the silica gel to the anhydrous sodium sulfate is 5; the normal hexane is used for leaching, and the ratio of the use volume to the mass of the silica gel in the purification column is 25mL/10g; in the elution, a mixed solution of ethyl acetate and n-hexane in a volume ratio of 1; the added solvent is isooctane, and the added volume is 5mL; the concentration specifically comprises the following steps: the mixture was first rotary evaporated to about 2mL at 32 ℃ and transferred to a centrifuge tube and concentrated to 1mL by nitrogen-blown evaporation.
And the fish sample to be detected is fresh fish or frozen fish, and if the sample is frozen fish, natural thawing is carried out before sample pretreatment, and the fish sample is returned to the room temperature.
Preferably, the step (1) is specifically: making a standard curve: respectively preparing a gradient concentration standard solution of each antioxidant standard product, calculating the actual concentration of each antioxidant standard product in the standard solution, detecting and analyzing the standard solution by using gas chromatography-tandem triple quadrupole mass spectrometry (GC-EI-MS/MS), determining the detected peak according to the mass spectrum result and the retention time of each antioxidant standard product, calculating the peak area of each component, taking 7-11 detection points, and performing linear regression on the actual concentration in the standard solution by using the peak area of each antioxidant standard product to obtain a standard curve.
The antioxidant standard substance is styrenated-N-phenylaniline
635 (BNS, benzamine, N-phenyl-, styrenated, CAS:68442-68-2; comprising 3 styrene amine (S-DPA) isomers and 2 stilbene diphenylamine (DS-DPA) isomers), N-phenylaniline and 2, 4-trimethylpentene
PS-30 (BNT, benzonamine, CAS #:68411-46-1; including tert-butyl/tert-octyldiphenylamine (C4/C8-DPA), tert-butyl-Diphenylamine (C4-DPA), tert-octyldiphenylamine (C8-DPA), diiso-tert-butyldiphenylamine (C4/C4-DPA), diisooctyldiphenylamine (C8/C8-DPA)), 2' -Dinaphthylamine (D2 NA, di-2-dinaphenylamine, #: 532-18-3), diphenylamine (DPA, diphenylamine, CAS #: 122-39-4), iminostilbene (DBA, 5h-dibenz [ b, f)]azepine, CAS #: 256-96-2), iminodibenzyl (IDB, imidibenzyl, CAS #: 494-19-9), benzhydrylamine (DPMA, diphenylmethyl amine, CAS #: 552-82-9), 1' -dinaphthylamine (D1 NA, di-1-dinaphtylamin, CAS #: 737-89-3), N-phenyl-1-naphthylamine (PNA, N-phenyl 1-1-naphthylamine, CAS # 90-30-2), N-phenyl o-phenylenediamine (ADPA, 2-Aminodiphenylamine, CAS # 534-85-0), diphenylamine (DPA, diphenylamine, CAS #: 122-39-4), di-sec-butyl-p-phenylenediamine (DBPDA, N, N ' -di-sec-butyl-1,4-phenylenediamine, CAS #: 101-96-2) or N- (1, 3-dimethyl) butyl-N ' -phenylenediamine (DMBPD, N- (1, 3-dimethylbutyl) -N ' -phenyl-1,4-phenylenediamine, CAS #: 793-24-8).
Preferably, the step (3) is specifically: detecting the sample liquid to be detected: and (3) detecting and analyzing the sample liquid to be detected obtained in the step (2) by using a gas chromatography-tandem triple quadrupole mass spectrometry (GC-EI-MS/MS), determining the detected peaks according to the mass spectrometry result and the retention time of each component, calculating to obtain the peak areas of each component, respectively substituting the peak areas of each component into respective standard curve equations to obtain the actual concentration of each component in the sample liquid to be detected, calculating to obtain the actual content of each component in the sample liquid to be detected, namely the actual content of each component in the fish sample to be detected in the step (2), and further calculating to obtain the content percentage of each component in the fish sample to be detected.
The detection analysis is carried out by using gas chromatography-tandem triple quadrupole mass spectrometry (GC-EI-MS/MS), wherein the gas chromatography uses an Agilent-7890A gas chromatography system, and the triple quadrupole mass spectrometry uses an Agilent 7000A triple quadrupole mass spectrometer; the gas chromatographic column is 30-m DB-5MS fused silica capillary column with inner diameter of 0.25mm and inner membrane of 0.25 μm (J & W Scientific, CA, USA); the temperature of a sample inlet is 300 ℃, and a1 mu L sample introduction non-shunting mode is adopted; the carrier gas is helium and nitrogen, and the purity is more than 99.99 percent; the flow rate of the helium gas is 2.25mL/min, and the flow rate of the nitrogen gas is 1.5mL/min; the flow rate of purging the isolation pad is 3mL/min; after 3min, the carrier gas flow rate is 20mL/min, and before 0.8min, the injection pulse pressure is 40psi; setting the flow rate of the purge gas flow to be 50mL/min at 1.2 min; the column temperature raising program is: keeping the temperature at 80 ℃ for 1min, then heating to 220 ℃ at the speed of 20 ℃/min, then heating to 250 ℃ at the speed of 5 ℃/min, and keeping the temperature for 1min; then raising the temperature to 300 ℃ at the speed of 20 ℃/min, keeping the temperature for 5min, and raising the temperature to 305 ℃ and keeping the temperature for 5min; the electron energy is 70eV, the ion source temperature is 300 ℃, and the mass spectrum MS1&2 temperature is 150 ℃; the mass spectrometry uses a scanning Multiple Reaction Monitoring (MRM) mode.
Advantageous effects
1. An instrumental analysis method for simultaneously detecting 20 antioxidants is established, and no literature report is available before;
2. establishing a method for detecting the antioxidant in the fish meat, wherein no literature report is available before;
3. the anhydrous sodium sulfate is ground with the sample, so that the water in the sample is removed, and the sample is uniformly mixed, so that the extraction effect is improved;
4. concentrated sulfuric acid is not used for sulfonation treatment, so that the damage and decomposition of a target object are avoided, and the interference is removed by using a gel chromatography and a silica gel column purification method;
5. compared with the liquid phase triple quadrupole method, the gas phase series triple quadrupole method has the advantages that the sensitivity is improved, and the separation effect is obviously better than that of the liquid phase.
Drawings
FIG. 1: the chemical structural formula of the target antioxidant;
FIG. 2: target antioxidant (100 ppb) mass spectrum (MRM);
FIG. 3: and detecting a mass-limiting spectrogram of the target antioxidant.
Detailed Description
Drugs and reagents
13C 12-Diphenylamine with a purity of 99% was purchased from Sigma-Aldrich (St. Lousi, MO, USA); antioxidant styrylized-N-phenylaniline
635 (BNS, benzamine, N-phenyl-, styrenated, CAS: 68442-68-2) and N-phenylaniline with 2, 4-trimethylpentene
PS-30 (BNT, benzamine, n-phenyl-, reaction products with 2, 4-trimethypentene, CAS #: 68411-46-1) available from Accustandard Inc. (New haven, CT, USA); 2,2' -Dinaphthylamine (D2 NA, di-2-Dinaphtylamine, CAS #: 532-18-3), diphenylamine (DPA, diphenylamine, CAS #: 122-39-4), iminostilbene (DBA, 5h-dibenz [ b, f [)]azepine, CAS #: 256-96-2), iminodibenzyl (IDB, iminodenzyl, CAS #: 494-19-9), benzhydrylamine (DPMA, diphenylmethyl amine, CAS #: 552-82-9), 1' -dinaphthylamine (D1 NA, di-1-dinaphtylamin, CAS #: 737-89-3), N-phenyl-1-naphthylamine (PNA, N-phenyl 1-1-naphthylamine, CAS #: 90-30-2), N-phenyl o-phenylenediamine (ADPA, 2-Aminodophenylamine, CAS #: 534-85-0), diphenylamine (DPA, diphenylamine, CAS #: 122-39-4), di-sec-butyl-p-phenylenediamine (DBPDA, N, N ' -di-sec-butyl-1,4-phenylenediamine, CAS #: 101-96-2) and N- (1, 3-dimethyl) butyl-N ' -phenyl-p-phenylenediamine (DMBPD, N- (1, 3-dimethyl) -N ' -phenyl-1,4-phenylenediamine, CAS #: 793-24-8) standards were obtained from Sigma Aldrich (Oakville, canada) and Tokyo chemical industries, inc. ((R)Portland, OR, USA), the above standard structural formula is shown in fig. 1.
Acetone (ACE, HPLC grade), hexane (HEX, HPLC grade), dichloromethane (DCM, HPLC grade) and isooctane (ISO, HPLC grade) were purchased from Fisher Scientific (Fair lawn, new Jersey, USA) and the ultrapure water used for the experiments (> 18M Ω -cm R) was prepared for Milli-Q pure water system (Millipore; billerica, MA, USA).
Examples
Preparing a sample solution to be detected:
the frozen fish meat was removed and thawed and returned to room temperature, and a 5g (fresh weight) sample of fish meat was taken, mixed with 20g of anhydrous sodium sulfate, ground into a fine powder and transferred to a 50mL glass centrifuge tube. Add 100. Mu.L of 1. Mu.g/mL 13 C 12 Internal standard of DPA, standing to equilibrate for 20min.
Adding 25mL (1/1, V/V) of acetone/n-hexane mixed solvent, oscillating for 30min, performing oscillation extraction, centrifuging for 15 min by using a centrifugal machine at the rotating speed of 3000r/min, transferring the supernatant to a 250mL flat-bottomed flask, repeating the extraction step for 2 times, combining the supernatant collected by 3 times of extraction to obtain an extract, adding 5mL of isooctane, and concentrating to about 1-2mL (nitrogen blowing instrument).
The extract was loaded onto a gel chromatography column (GPC, 30mm × 10mm, biotead S-X3), the target contaminant was eluted with a 50mL solution of ethyl acetate and cyclohexane (1.
Filling 10g of 5% inactivated silica gel and 2g of anhydrous sodium sulfate into a purification column, loading an extraction liquid purified by GPC into the purification column for further purification, and leaching interferents by using 25mL of n-hexane; then eluting the target substance by using a 45mL mixed solvent of ethyl acetate and n-hexane (1.
Preparation of standard solutions:
taking the compounds in the table 1, preparing a gradient concentration standard solution of each antioxidant standard, and calculating the actual concentration of each antioxidant standard in the standard solution.
Making a standard curve:
the measurements were performed using an Agilent-7890A gas chromatography system with an Agilent 7000A triple quadruple mass spectrometer (GC-EI-MS/MS). The gas chromatography column used was a 30-m DB-5MS fused silica capillary column with an internal diameter of 0.25mm and an internal membrane of 0.25 μm (J & W Scientific, CA, USA). The sample inlet is 300 ℃, and a1 mu L sample inlet non-shunting mode is adopted. The carrier gases were helium and nitrogen with a purity of >99.99%. The flow rate of helium was 2.25mL/min and that of nitrogen was 1.5mL/min. The flow rate was set at 1.2mL/min and the septum purge flow rate was set at 3mL/min. After 3min, the protective gas is 20mL/min, and before 0.8min, the injection pulse pressure is 40psi; at 1.2min, the purge flow rate was set at 50mL/min. The column temperature raising program is: keeping the temperature at 80 ℃ for 1min, raising the temperature to 220 ℃ at a speed of 20 ℃/min, then raising the temperature to 250 ℃ at a speed of 5 ℃/min, and keeping the temperature for 1min; then, the temperature is raised to 300 ℃ at a speed of 20 ℃/min, and then the temperature is maintained for 5min, and the temperature is raised to 305 ℃ and then the temperature is maintained for 5min. The electron energy was 70eV, the ion source temperature was 300 ℃ and the mass spectra MS1&2 temperatures were 150 ℃. The mass spectra were obtained in a scanning Multiple Reaction Monitoring (MRM) mode, where MRM monitors ion pairs, retention time and collision energy as shown in table 1. The spectrum of the standard is shown in FIG. 2.
Detecting and analyzing the standard solution by using a gas chromatography-tandem triple quadrupole mass spectrometry (GC-EI-MS/MS), determining the detected peak according to the mass spectrometry result and the retention time of each antioxidant standard, calculating the peak area of each component, taking 7-11 detection points, and performing linear regression on the actual concentration in the standard solution by using the peak area of each antioxidant standard to obtain a standard curve.
Actual detection:
detecting and analyzing the sample liquid to be detected by using the same chromatographic and mass spectrometric conditions and using gas chromatography-tandem triple quadrupole mass spectrometry (GC-EI-MS/MS), determining the detected peaks according to the mass spectrometric result and the retention time of each component, then calculating to obtain peak areas of each component, respectively substituting the peak areas of each component into respective standard curve equations to obtain the actual concentration of each component in the sample liquid to be detected, calculating to obtain the actual content of each component in the sample liquid to be detected, namely the actual content of each component in the fish sample to be detected according to the mass = concentration x volume, and further calculating to obtain the content percentage of each component in the fish sample to be detected.
Table 1: GC-MS/MS detection condition of target pollutant
Method detection limit
The detection limit of the target is an index for representing the sensitivity, and the lower the detection limit, the better the sensitivity. The limit of detection (LOD) of the instrument is signal-to-noise ratio (S/N) >3, and the limit of quantitation (LOQ) of the instrument is determined by signal-to-noise ratio (S/N) > 10. The LOD and LOQ values of the target compounds DPA, D2NA, D1NA, PNA, IDB, DBA, DPMA and DBPDA are 0.05 and 0.15ng/mL, respectively, the LOD and LOQ values of ADPA are 0.5 and 1.5ng/mL, respectively, the LOD and LOQ values of DMBPD are 0.1 and 0.3ng/mL, respectively, the LOD values of C4-DPA, C8/C8-DPA and S-DPA1 are 0.02ng/mL, the LOQ values are 0.06ng/mL, the LOD values of C4/C8-DPA and S-DPA2 are 0.04ng/mL, the LOQ values are 0.12ng/mL, the LOD values of DS-DPA1 and DS-DPA3 are 0.07/mL, the LOQ values are 0.21ng/mL, the LOD values of C8-DPA and C4/C4-DPA are 0.1ng/mL, the LOD values of 0.1ng/mL, the LOQ values of DPA are 0.08, and the LOQ values are 0.24ng/mL. (FIG. 3)
Based on the sample size of 5g of fish meat, the quantitative limit of the whole pretreatment method is calculated: LOQ of DPA, D2NA, D1NA, PNA, IDB, DBA, DPMA, DBPDA and DS-DPA2 is 0.05ng/g (wet weight), LOQ of DMBPD, C8-DPA and C4/C4-DPA is 0.06ng/g (wet weight), LOQ of C4-DPA, C8/C8-DPA and S-DPA1 is 0.01ng/g (wet weight); LOQ of C4/C8-DPA and S-DPA2 was 0.02ng/g (wet weight), and LOQ of DS-DPA1 and DS-DPA3 was 0.04ng/g (wet weight).
Method recovery
The recovery rate can be evaluated for the accuracy of the measurement method, and the sample measurement method should have a stable recovery rate within a specified range. Target contaminants of mass 100 and 500ng (n = 6) were added to the homogenized 5g of fish meat, respectively. The recovery of these compounds through the entire analytical method ranged from 58.3% to 97.6% with a Relative Standard Deviation (RSD) of between 3.02 and 12.4%. If the analyte concentration in the sample is outside the standard curve range, the extract is diluted or concentrated to within the calibration range of the instrument and re-analyzed.
Practical application
The method is verified by a blank experiment, a blank labeling experiment, a matrix labeling experiment and an actual sample experiment. The method is suitable for analyzing fish samples, and the total concentration of antioxidant in the actual samples is 0.53-102ng/g (wet weight), wherein ADPA, DBPDA and DS-DPA1,2,3 are not detected in all samples. Internal standard in sample: ( 13 C 12 Recovery of-DPA) averaged 73% and RSD was 4.1%.
Claims (3)
1. A method for detecting antioxidants in fish meat, which is characterized by comprising the following steps: the method comprises the following steps:
(1) Making a standard curve: respectively preparing a gradient concentration standard solution of each antioxidant standard substance, respectively using gas chromatography-tandem triple quadrupole mass spectrometry to detect and analyze, calculating the area of each component, and performing linear regression on the actual concentration of each component in the standard solution by using the peak area of each component to obtain a standard curve; the antioxidant standards are shown in the following table:
(2) Preparing a sample solution to be detected: 1) Fish meat pretreatment: taking fish sample to be detected, adding anhydrous sodium sulfate, mixing, and grindingGrinding into fine powder, adding internal standard, and mixing; the mass ratio of the fish sample to be detected to the anhydrous sodium sulfate is 1; the internal standard is 13 C 12 DPA, the mass ratio of the added mass of the internal standard to the fish sample to be detected is 0.1 multiplied by 10 -6 5, preparing a mixture; finally, uniformly mixing, standing and balancing for 20min;
2) And (3) extraction: adding a solvent into the pretreated fish sample for extraction, and concentrating the extract into a crude sample solution; the extraction is specifically as follows: adding a solvent, oscillating, collecting extract liquor, repeating the extraction step for 2 times, combining the extract liquor obtained in the three times, adding isooctane, and concentrating, wherein the used solvent is an acetone/n-hexane mixed solvent, and the volume ratio of acetone/n-hexane in the mixed solvent is 1; the ratio of the volume of the solvent added in each extraction to the mass of the fish sample to be detected is 5mL; the shaking time is 30min each time; after shaking extraction, centrifuging for 15 minutes by using a centrifugal machine at the rotating speed of 3000r/min, collecting the upper layer, then repeating the extraction step for 2 times, and combining the upper layers obtained by three times of extraction and collection to obtain extract liquor; the volume of the added isooctane is 1/5 of the volume of the solvent added each time in the extraction process; concentrating, wherein the volume after concentration is 1-2mL;
3) Purifying: purifying the crude sample solution by a gel chromatographic column, performing rotary evaporation, concentrating by a nitrogen blowing instrument, and adding isooctane to obtain a primary purified sample solution; the gel chromatographic column purification specifically comprises the following steps: loading the crude sample liquid to a gel chromatographic column of 30mm multiplied by 10mm, biobead S-X3, eluting the target pollutant by using mixed liquid of ethyl acetate and cyclohexane with the volume ratio of 1; the volume of the added isooctane is 1-2mL;
4) Further purification: loading the primarily purified sample liquid to a purification column filled with silica gel and anhydrous sodium sulfate, eluting, adding a solvent, and concentrating to obtain a sample liquid to be detected; the filling column material of the purification column is 5% of inactivated silica gel and anhydrous sodium sulfate, the mass ratio of the silica gel to the anhydrous sodium sulfate is 5; the normal hexane is used for leaching, and the ratio of the use volume to the mass of the silica gel in the purification column is 25mL/10g; in the elution, a mixed solution of ethyl acetate and n-hexane in a volume ratio of 1; the added solvent is isooctane, and the added volume is 5mL; the concentration specifically comprises the following steps: rotationally evaporating to about 2mL at 32 ℃, transferring to a centrifuge tube, and carrying out nitrogen blowing concentration to 1 mL;
(3) Detecting and analyzing the sample liquid to be detected obtained in the step (2) by using gas chromatography-tandem triple quadrupole mass spectrometry, determining the detected peak according to the mass spectrometry result and the retention time of each component, calculating to obtain the peak area of each component, substituting the peak area of each component into respective standard curve equation to obtain the actual concentration of each component in the sample liquid to be detected, calculating to obtain the actual content of each component in the sample liquid to be detected according to the mass = concentration by volume, namely calculating the actual content of each component in the fish sample to be detected in the step (2), and further calculating to obtain the content percentage of each component in the fish sample to be detected;
the above steps are performed in the order of steps (1), (2) and (3), or in the order of steps (2), (1) and (3).
2. The method for detecting antioxidants in fish meat according to claim 1, characterized in that: the step (1) is specifically as follows: making a standard curve: respectively preparing a gradient concentration standard solution of each antioxidant standard product, calculating the actual concentration of each antioxidant standard product in the standard solution, detecting and analyzing the standard solution by using gas chromatography-tandem triple quadrupole mass spectrometry, determining the detected peak according to the mass spectrum result and the retention time of each antioxidant standard product, calculating the peak area of each component, taking 7-11 detection points, and performing linear regression on the actual concentration of each antioxidant standard product in the standard solution by using the peak area of each antioxidant standard product to obtain a standard curve.
3. The method for detecting antioxidants in fish meat according to claim 1, characterized in that: the gas chromatography-tandem triple quadrupole mass spectrometry combination is used, wherein an Agilent-7890A gas chromatography system is used for gas chromatography, and an Agilent 7000A triple quadrupole mass spectrometer is used for triple quadrupole mass spectrometry; the gas chromatographic column is a 30-m DB-5MS fused quartz capillary column of J & W Scientific, CA, USA, the inner diameter is 0.25mm, and the inner membrane is 0.25μm; the temperature of a sample inlet is 300 ℃, and a1 mu L sample introduction non-shunting mode is adopted; the carrier gas is helium and nitrogen, and the purity is more than 99.99%; the flow rate of the helium gas is 2.25mL/min, and the flow rate of the nitrogen gas is 1.5mL/min; the flow rate of purging the isolation pad is 3mL/min; after 3min, the carrier gas flow rate is 20mL/min, and before 0.8min, the injection pulse pressure is 40psi; setting the flow rate of the purge gas flow to be 50mL/min at 1.2 min; the column temperature raising program is: 80. keeping at 1min, heating to 220 deg.C at 20 deg.C/min, heating to 250 deg.C at 5 deg.C/min, and keeping for 1min; then raising the temperature to 300 ℃ at the speed of 20 ℃/min, keeping the temperature for 5min, and raising the temperature to 305 ℃ and keeping the temperature for 5min; the electron energy is 70eV, the ion source temperature is 300 ℃, and the mass spectrum MS1&2 temperature is 150 ℃; the mass spectrum adopts a scanning multi-reaction monitoring mode.
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