CN110220997A - A kind of method of antioxidant in the detection flesh of fish - Google Patents
A kind of method of antioxidant in the detection flesh of fish Download PDFInfo
- Publication number
- CN110220997A CN110220997A CN201910610811.6A CN201910610811A CN110220997A CN 110220997 A CN110220997 A CN 110220997A CN 201910610811 A CN201910610811 A CN 201910610811A CN 110220997 A CN110220997 A CN 110220997A
- Authority
- CN
- China
- Prior art keywords
- flesh
- fish
- antioxidant
- sample
- cas
- 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
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
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)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a kind of methods of antioxidant in detection flesh of fish, it is related to pollutants in food detection method, it is developed for the detection method of phenyl amines antioxidant in the flesh of fish, pass through the processes such as extracting and purifying instrument analysis, the concentration of phenyl amines antioxidant in the flesh of fish is detected, method provided by the invention is realized by the following means: production antioxidant standard curve；Prepare analyte sample fluid: flesh of fish sample to be measured is preprocessed, extraction, purifies and further purifies, and obtains analyte sample fluid；Analyte sample fluid is tested and analyzed using gas-chromatography-series connection triple level four bars mass spectrometries (GC-EI-MS/MS), percentage composition of each ingredient in flesh of fish sample to be measured is calculated.Above method detection limit is low, and the rate of recovery reaches standard, can be used for that antioxidant in the practical flesh of fish is qualitative and quantitative detection.
Description
Technical field
The present invention relates to pollutants in food detection methods, and in particular to a method of antioxidant in the detection flesh of fish.
Background technique
Antioxidant (Antioxidants) is the substance for blocking, restraining or delaying polymer oxidation or autoxidation.It
It can capture and neutralize various free free radicals, such as O2-, OH and HO2Etc., thus mitigate or remove to polymer and
The damage that human body generates.Antioxidant can improve the oxidation stability of some oil products such as gasoline, diesel oil significantly and prolong
Long polymer uses the time.At the end of the 19th century to early 20th century, antioxidant is widely used for oil product, rubber in relevant industries
The quality-improving in the fields such as glue, plastics.Such as be used to avoid the formation of the vulcanization of rubber, metal erosion, fuel polymerize to be formed it is interior
Combustion engine accumulates dirt etc..P-phenylenediamine and β-aniline work in amine substance are just used in 19 century 70 Bogge
For the oxidative degradation agent of rubber product, so that rubber obtained great extension using the time, but until 20 years 20th century
The concept in generation, antioxidant is just contemplated by Moureon for the first time.In subsequent development in more than 100 years, new antioxidant
Kind continuously emerges, and promotes the development of modern industry, such as the development of new material, automobile.
Nineteen thirty-seven, the in the world antioxidant 2 of first hindered phenol structure, 6- di-tert-butyl-4-methy phenol (BHT) quilt
It was found that BHT has also been developed at the beginning of the eighties in last century in China.It is shown in the investigation result of the last century 80's Mo, China
Plastics antioxidant productive consumption is less than 1 kiloton, more much lower than some western countries.By 2002, Chinese Plastics antioxygen
The aggregate consumption of agent is more than 20,000 tons, in the late 1980s close to the U.S., West Europe and other countries.Currently, China is certainly
The antioxidant kind and quality of main research and development can satisfy the demand of Chinese Plastics and fuel industry substantially, on this basis, in
State can all export some antioxidant products every year.Most common three kinds of antioxidants are mainly phenol antioxidant in the market,
Phosphorus antioxidant and amine antioxidants.
As antioxidant is using more and more extensive, environmental pollution feature is constantly changing, part antioxygen
Agent has refractory organics and bioconcentration, such as phenyl amines antioxidant.These target contaminants enter natural water body,
And start gradually to accumulate, increase the original concentration of trace contaminant by several times, leads to microorganism, animal and the plant in natural water body
Object generates resistance.There are some antioxidants to be difficult to be degraded in nature, it was reported that antioxidant is eventually with food chain
It enters in human body, so that the frequency of human carcinogen and cause endocrine disturbance increases.Exploitation and people with new drug
Improvement of living standard, the more flourishing city of economy will generate more antioxidants.But at present for antioxidant
Correlative study such as aniline category matter and its derivative analyte detection is less, and without data and database can consult these substances
Content in existing environment water, therefore the content in the detection method about antioxidant and environment just becomes primary study
Object.With the raising of scientific and technological level, more and more chemicals are invented and are applied, but its toxicological effect it is not yet clear and
Not by strict control, these chemicals are caused largely to enter natural environment, including atmosphere, soil, surface water system, and then influence
Wildlife (fish, shrimp and birds etc.).Currently, not yet effectively for the effective detection method of antioxidant composition in the flesh of fish
Report, more have no while detecting the detection method of a variety of antioxidant compositions in the flesh of fish.
Summary of the invention
The present invention is developed for the detection method of phenyl amines antioxidant in the flesh of fish, is analyzed by extracting and purifying instrument
Etc. processes, detect the flesh of fish in phenyl amines antioxidant concentration.
The method of antioxidant in the detection flesh of fish provided by the invention, comprising the following steps:
(1) it makes standard curve: the gradient concentration standard solution of every kind of antioxidant standard items is respectively configured, uses respectively
The triple level four bars mass spectrometries (GC-EI-MS/MS) of gas-chromatography-series connection test and analyze, and calculate the area of each ingredient, Yi Gecheng
Swarming area carries out linear regression to actual concentrations of each ingredient in standard solution and obtains standard curve；
(2) prepare analyte sample fluid: flesh of fish sample to be measured is preprocessed, extraction, purifies and further purifies, and obtains to be measured
Sample liquid；
(3) using the triple level four bars mass spectrometries (GC-EI-MS/MS) of gas-chromatography-series connection to be measured obtained by step (2)
Sample liquid is tested and analyzed, and each Component peak area is calculated, and it is bent that the peak area of each ingredient is substituted into respective standard respectively
Line equation obtains actual concentrations of each ingredient in analyte sample fluid, further calculates to obtain each ingredient in flesh of fish sample to be measured
In percentage composition in flesh of fish sample to be measured of actual content, each ingredient.
Above step, according to step (1), (2), (3) sequence carry out, or according to step (2), (1), (3) sequence into
Row.
Preferably, above-mentioned steps (2) also include the following specific steps:
1) flesh of fish pre-processes: taking flesh of fish sample to be measured, anhydrous sodium sulfate mixing is added, is ground into fine powder, it is mixed that internal standard is added
It closes uniform；
2) extract: solvent extraction is added in pretreated flesh of fish sample, and extract liquor concentration is crude samples liquid；
3) purify: crude samples liquid is added isooctane, obtains just after gel chromatography column purification, rotary evaporation, nitrogen evaporator concentration
Step purification sample liquid；
4) further purification: the decontaminating column of preliminary purification sample liquid loading to silica gel and anhydrous sodium sulfate filling, after elution
Elution is added solvent, is concentrated to get analyte sample fluid.
The mass ratio of the step 1) flesh of fish sample to be measured and anhydrous sodium sulfate is 1:4；It is designated as in described13C12- DPA, it is interior
It is 0.1 × 10 that quality and the mass ratio of flesh of fish sample to be measured, which is added, in mark-6:5；Balance 20min is finally stood after mixing.
Step 2) the extraction specifically: solvent is added, extract liquor is collected in concussion, is repeated above step 2 times, merges three
Isooctane, concentration is added in secondary extract liquor.
Solvent for use is acetone/n-hexane mixed solvent, and in the mixed solvent acetone/n-hexane volume ratio is 1:1；Extraction every time
The ratio of the solvent volume and flesh of fish sample quality to be measured that take addition is 5mL:1g；The concussion time is 30min every time；Extract through concussion
It after taking, is centrifuged 15 minutes, revolving speed 3000r/min using centrifuge, collects upper layer, repeatedly extraction step 2 times later merge three
The upper layer that secondary extraction is collected is extract liquor；The volume that isooctane is added is that the 1/5 of solvent volume is added every time in extraction process；
The concentration, the volume after concentration are 1-2mL.
Step 3) the gel chromatography column purification specifically: crude samples liquid loading to gel chromatographic columns (GPC, 30mm ×
10mm, Biobead S-X3), the mixed liquor elution target contaminant for being 1:1 with cyclohexane volume ratio with ethyl acetate is simultaneously collected
Eluent.The volume that isooctane is added is 1-2mL.
Step 4) the decontaminating column, filling column material are the silica gel and anhydrous sodium sulfate of 5% inactivation, silica gel and anhydrous sodium sulfate
Mass ratio be 5:1, anhydrous sodium sulfate loadings are 2g in decontaminating column；The elution uses volume and purification using n-hexane
The ratio of the quality of silica gel is 25mL/10g in column；The elution, the mixing for the use of ethyl acetate and n-hexane volume ratio being 1:1
Liquid, the ratio using the quality of silica gel in volume and decontaminating column are 45mL/10g；The solvent of the addition is isooctane, addition
Volume is 5mL；The concentration, specifically: first rotary evaporation is transferred in centrifuge tube, nitrogen blows dense to about 2mL under the conditions of 32 DEG C
It is reduced to 1mL.
The flesh of fish sample to be measured is fresh fish or the freezing flesh of fish, if sample is the freezing flesh of fish, before carrying out sample pretreatment
It carries out natural thaw and returns back to room temperature.
Preferably, above-mentioned steps (1) specifically: the ladder of every kind of antioxidant standard items production standard curve: is respectively configured
Concentration standard solution is spent, actual concentrations of each antioxidant standard items in its standard solution is calculated, uses gas-chromatography-string
Join triple level four bars mass spectrometries (GC-EI-MS/MS) to test and analyze standard solution, according to mass spectral results and each antioxygen
The retention time of agent standard items is qualitative to the progress of detection peak, calculates the peak area of each ingredient, 7~11 test points is taken, with each
Antioxidant standard items peak area carries out linear regression to the actual concentrations in its standard solution and obtains standard curve.
The antioxidant standard items are styrenyl-N- phenylaniline635(BNS,
benzenamine,n-phenyl-,styrenated,CAS:68442-68-2；Including 3 kinds of styrylamine (S-DPA) isomers
With 2 kinds of talan diphenylamines (DS-DPA) isomers), N- phenylaniline and 2,4,4- trimethylpentene
PS-30(BNT,benzenamine,n-phenyl-,reaction products with 2,4,4-trimethypentene,
CAS#:68411-46-1；Including tert-butyl/t-octyl diphenylamines (C4/C8-DPA), tert-butyl-diphenylamines (C4-DPA), tertiary pungent
Base diphenylamines (C8-DPA), two different tert-butyl diphenylamines (C4/C4-DPA), di-iso-octyldiphenylamine (C8/C8-DPA)), 2,2'-
Dinaphthylamine (D2NA, di-2-Dinaphthylamine, CAS#:532-18-3), diphenylamines (DPA, Diphenylamine,
CAS#:122-39-4), iminostilbene (DBA, 5h-dibenz [b, f] azepine, CAS#:256-96-2), iminodibenzyl
(IDB, Iminodibenzyl, CAS#:494-19-9), benzhydrylamine (DPMA, Diphenylmethylamine, CAS#:552-
82-9), 1,1'- dinaphthylamine (D1NA, di-1-Dinaphthylamin, CAS#:737-89-3), N- phenyl-1-naphthylamine (PNA,
N-Pheny1-1-naphthylamine, CAS#:90-30-2), N- phenyl o-phenylenediamine (ADPA, 2-
Aminodiphenylamine, CAS#:534-85-0), diphenylamines (DPA, Diphenylamine, CAS#:122-39-4), two
Secondary-butyl-p-phenylenediamine (DBPDA, N, N'-di-sec-butyl-1,4-phenylenediamine, CAS#:101-96-2) or
N- (1,3- dimethyl) butyl-N'- diphenyl-para-phenylene diamine (DMBPD, N- (1,3-dimethylbutyl)-N'-phenyl-1,4-
Phenylenediamine, CAS#:793-24-8).
Preferably, above-mentioned steps (3) specifically: analyte sample fluid detection: use the triple level four bars matter of gas-chromatography-series connection
Spectrum combination (GC-EI-MS/MS) tests and analyzes analyte sample fluid obtained by step (2), according to mass spectral results and respectively at code insurance
It stays the time to carry out detection peak qualitative, each Component peak area is then calculated, the peak area of each ingredient is substituted into respectively respectively
Calibration curve equation, obtain actual concentrations of each ingredient in analyte sample fluid, according to quality=concentration * volume, calculate
To actual content of each ingredient in analyte sample fluid, i.e., the actual content of to be measured flesh of fish sample of each ingredient in step (2),
It further calculates to obtain percentage composition of each ingredient in flesh of fish sample to be measured.
The triple level four bars mass spectrometries (GC-EI-MS/MS) of the use gas-chromatography-series connection test and analyze, wherein gas phase
Chromatography uses Agilent-7890A gas chromatography system, and triple level four bars mass spectrums use Agilent 7000A triple quadrupole bar mass spectrum
Instrument；Gas chromatographic column is 30-m DB-5MS Fused-silica capillary column, internal diameter 0.25mm, 0.25 μm of (J&W of inner membrance
Scientific,CA,USA)；Injector temperature is 300 DEG C, using 1 μ L sample introduction not shunt mode；Carrier gas be helium and nitrogen,
Purity > 99.99%；Helium gas flow is 2.25mL/min, nitrogen 1.5mL/min；It is 3mL/min that dottle pin, which purges flow velocity,；3min
Afterwards, before flow rate of carrier gas 20mL/min, 0.8min, injection pulse pressure is 40psi；In 1.2min, purge stream flow velocity is set
For 50mL/min；Column temperature temperature program are as follows: after 80 DEG C of holding 1min, be raised to 220 DEG C with the speed of 20 DEG C/min, then with 5 DEG C/
The speed of min is raised to 250 DEG C, and keeps 1min；Then after being raised to 300 DEG C with the speed of 20 DEG C/min, 5min is kept, is raised to
5min is kept after 305 DEG C；Electron energy is 70eV, and ion source temperature is 300 DEG C, and mass spectrum MS1&2 temperature is 150 DEG C；Mass spectrum is adopted
It is scan-type multiple-reaction monitoring (multiple reaction monitoring, MRM) mode.
Beneficial effect
1. the instrument analytical method of 20 kinds of antioxidants is established while detecting, before without document report；
2. the method for establishing antioxidant in the detection flesh of fish, before without document report；
3. ground using anhydrous sodium sulfate and sample, that is, eliminating the moisture in sample and also mixing sample mentions effect of extracting
It rises；
4. not carrying out sulfonation processing using the concentrated sulfuric acid, avoids the destruction of object from decomposing, utilize gel chromatography and silica gel
The purification method of column removes interference；
The method of the triple level four bars level four bars method sensitivity enhancements more triple than liquid phase 5. gas phase is connected, and separating effect
Significantly better than liquid phase.
Detailed description of the invention
Fig. 1: object antioxidant chemical structural formula；
Fig. 2: object antioxidant (100ppb) mass spectrogram (MRM)；
Fig. 3: object antioxidant detection limit mass spectrogram.
Specific embodiment
Drug and reagent
The 13C12- diphenylamines that purity is 99% is purchased from Sigma-Aldrich (St.Lousi, MO, USA)；Antioxidant benzene
Vinylated-N- phenylaniline635(BNS,benzenamine,n-phenyl-,styrenated,CAS:
68442-68-2) and N- phenylaniline and 2,4,4- trimethylpentene PS-30(BNT,benzenamine,n-
Phenyl-, reaction products with 2,4,4-trimethypentene, CAS#:68411-46-1) it is purchased from
Accustandard Inc.(New haven,CT,USA)；2,2'- dinaphthylamines (D2NA, di-2-Dinaphthylamine,
CAS#:532-18-3), diphenylamines (DPA, Diphenylamine, CAS#:122-39-4), iminostilbene (DBA, 5h-dibenz
[b, f] azepine, CAS#:256-96-2), iminodibenzyl (IDB, Iminodibenzyl, CAS#:494-19-9), hexichol
Methylamine (DPMA, Diphenylmethylamine, CAS#:552-82-9), 1,1'- dinaphthylamine (D1NA, di-1-
Dinaphthylamin, CAS#:737-89-3), N- phenyl-1-naphthylamine (PNA, N-Pheny1-1-naphthylamine,
CAS#:90-30-2), N- phenyl o-phenylenediamine (ADPA, 2-Aminodiphenylamine, CAS#:534-85-0), diphenylamines
(DPA, Diphenylamine, CAS#:122-39-4), di-sec-butyl-p-phenyl enediamine (DBPDA, N, N'-di-sec-butyl-1,
4-phenylenediamine, CAS#:101-96-2) and N- (1,3- dimethyl) butyl-N'- diphenyl-para-phenylene diamine (DMBPD,
N- (1,3-dimethylbutyl)-N'-phenyl-1,4-phenylenediamine, CAS#:793-24-8) standard items purchase
It is above from Sigma-Aldrich (Oakville, Canada) and Tokyo chemical conversion industry company (Portland, OR, USA)
Standard items structural formula is as shown in Figure 1.
Acetone (ACE, HPLC grades), hexane (HEX, HPLC grades), methylene chloride (DCM, HPLC grades) and isooctane (ISO,
HPLC grades) it is purchased from Fisher Scientific (Fair lawn, New Jersey, USA), test ultrapure water (> 18M Ω-used
Cm R) it is that Milli-Q pure water system prepares (Millipore；Billerica, MA, USA).
Embodiment
Prepare analyte sample fluid:
It takes out the freezing flesh of fish to thaw, restores to room temperature, take 5g (fresh weight) flesh of fish sample, mixed with 20g anhydrous sodium sulfate, and
It is ground into fine powder, is transferred to 50mL glass centrifuge tube.The 1 μ g/mL of 100 μ L is added13C12- DPA internal standard compound stands balance
20min。
Add acetone/n-hexane mixed solvent 25mL (1/1, V/V), vibrate 30min, after concussion extraction, using from
Scheming is centrifuged 15 minutes, revolving speed 3000r/min, is shifted upper liquid to 250mL boiling flask, is repeated extraction step 2 later
It is secondary, merge 3 times and extract collected upper liquid, is extract liquor, 5mL isooctane is added, is concentrated into about 1-2mL (nitrogen evaporator).
By extract liquor loading to gel chromatographic columns (GPC, 30mm × 10mm, Biobead S-X3), with 50mL ethyl acetate
Target contaminant is eluted with hexamethylene (1:1V:V) solution and collects eluent with 250mL boiling flask, through Rotary Evaporators, nitrogen
It blows instrument concentration and solvent is replaced into isooctane, volume about 1-2mL, to purify in next step.
Silica gel 10g, the anhydrous sodium sulfate 2g of 5% inactivation is taken to fill decontaminating column, through the purified extract liquor loading of GPC to net
Change column further to purify, elutes chaff interferent with the n-hexane of 25mL；Then the ethyl acetate and n-hexane mixed solvent of 45mL are used
(1:1V:V) elution object simultaneously collects leacheate, and after 5mL isooctane is added, rotary evaporation turns to about 2mL under the conditions of 32 DEG C
It moves in centrifuge tube, nitrogen, which is blown, is concentrated into 1mL, obtains analyte sample fluid, is finally transferred in brown chromatogram bottle and deposits in -20 DEG C of refrigerators
Middle preservation, stand-by GC-MS/MS are detected.
Prepare standard solution:
The compound in table 1 is taken, the gradient concentration standard solution of every kind of antioxidant standard items is respectively configured, is calculated each
Actual concentrations of the antioxidant standard items in its standard solution.
Make standard curve:
Use Agilent-7890A gas chromatography system and the triple quadruple mass spectrographs (GC-EI-MS/MS) of Agilent 7000A
It is measured.Gas chromatographic column uses 30-m DB-5MS Fused-silica capillary column, internal diameter 0.25mm, and 0.25 μm of inner membrance
(J&W Scientific,CA,USA).300 DEG C of injection port, using 1 μ L sample introduction not shunt mode.Carrier gas be helium and nitrogen, it is pure
Degree > 99.99%.Helium gas flow is 2.25mL/min, nitrogen 1.5mL/min.Flow velocity is set as 1.2mL/min, dottle pin purges
Flow velocity is 3mL/min.After 3min, protection gas is 20mL/min, and before 0.8min, injection pulse pressure is 40psi；In 1.2min,
Purge stream flow velocity is set as 50mL/min.Column temperature temperature program are as follows: after 80 DEG C of holding 1min, be raised to the speed of 20 DEG C/min
220 DEG C, 250 DEG C then are raised to the speed of 5 DEG C/min, and keep 1min；Then 300 DEG C are raised to the speed of 20 DEG C/min
Afterwards, 5min is kept, keeps 5min after being raised to 305 DEG C.Electron energy is 70eV, and ion source temperature is 300 DEG C, mass spectrum MS1&2 temperature
Degree is 150 DEG C.Mass spectrum using scan-type multiple-reaction monitoring (multiple reaction monitoring, MRM) mode,
It is as shown in table 1 that MRM monitors ion pair, retention time and impact energy.Standard items spectrogram is as shown in Figure 2.
Detection point is carried out to standard solution using gas-chromatography-series connection triple level four bars mass spectrometries (GC-EI-MS/MS)
Analysis, it is qualitative to the progress of detection peak according to the retention time of mass spectral results and each antioxidant standard items, calculate the peak face of each ingredient
Product, takes 7~11 test points, is carried out with each antioxidant standard items peak area to the actual concentrations in its standard solution linear
It returns and obtains standard curve.
It is actually detected:
Using same chromatography, Mass Spectrometry Conditions, with the triple level four bars mass spectrometry (GC-EI-MS/ of gas-chromatography-series connection
MS) analyte sample fluid is tested and analyzed, it is qualitative to the progress of detection peak according to mass spectral results and each ingredient retention time, then
Each Component peak area is calculated, the peak area of each ingredient is substituted into respective calibration curve equation respectively, obtains each ingredient and exists
Reality of each ingredient in analyte sample fluid is calculated according to quality=concentration * volume in actual concentrations in analyte sample fluid
Content, i.e., each ingredient further calculate to obtain each ingredient in flesh of fish sample to be measured in the actual content of flesh of fish sample to be measured
Percentage composition.
Table 1: the GC-MS/MS testing conditions of target contaminant
Method detection limit
Object detection limit is a kind of index for characterizing sensitivity, and detection limit is lower, and expression sensitivity is better.The inspection of instrument
Surveying limit (LOD) is signal-noise ratio (S/N) > 3, and the quantitative limit (LOQ) of instrument is determined by signal-to-noise ratio (S/N) > 10.Target chemical combination
LOD the and LOQ value of object DPA, D2NA, D1NA, PNA, IDB, DBA, DPMA and DBPDA are respectively 0.05 and 0.15ng/mL, ADPA
LOD and LOQ value be respectively 0.5 and 1.5ng/mL, LOD the and LOQ value of DMBPD is respectively 0.1 and 0.3ng/mL, C4-DPA,
The LOD of C8/C8-DPA and S-DPA1 is that 0.02ng/mL, LOQ are limited to the LOD of 0.06ng/mL, C4/C8-DPA and S-DPA2 and are
The LOD of 0.04ng/mL, LOQ 0.12ng/mL, DS-DPA1 and DS-DPA3 are 0.07ng/mL, LOQ 0.21ng/mL, C8-
The LOD of DPA and C4/C4-DPA is 0.1ng/mL, LOQ 0.3ng/mL, and the LOD of DS-DPA2 is that 0.08ng/mL, LOQ are
0.24ng/mL.(Fig. 3)
Based on the sample size of the 5g flesh of fish, calculate the quantitative limit of entire preprocess method: DPA, D2NA, D1NA, PNA,
The LOQ of IDB, DBA, DPMA, DBPDA and DS-DPA2 are 0.05ng/g (weight in wet base), the LOQ of DMBPD, C8-DPA and C4/C4-DPA
Value is 0.06ng/g (weight in wet base), C4-DPA, the LOQ of C8/C8-DPA and S-DPA1 be 0.01ng/g (weight in wet base)；C4/C8-DPA and
The LOQ of S-DPA2 is 0.02ng/g (weight in wet base), and the LOQ of DS-DPA1 and DS-DPA3 are 0.04ng/g (weight in wet base).
The method rate of recovery
The rate of recovery can evaluate the accuracy of measuring method, and sample measuring method should have stable within the limits prescribed
The rate of recovery.The target contaminant that quality is 100 and 500ng (n=6) is added in the 5g flesh of fish after homogenate respectively.These are changed
Object is closed by the rate of recovery range of entire analysis method in 58.3%-97.6%, relative standard deviation (RSD) is in 3.02-
Between 12.4%.If the analyte concentration in sample exceeds standard curve range, extract liquor is diluted or is concentrated into instrument
Calibration range in and reanalyse.
Practical application
Pass through blank assay, the experiment of blank mark-on, the experiment of matrix mark-on and actual sample experimental verification.This method is suitable for
The analysis of flesh of fish sample, the total concentration of antioxidant distinguishes 0.53-102ng/g (weight in wet base) in actual sample, wherein ADPA,
DBPDA and DS-DPA1,2,3 are not detected in all samples.In sample internal standard compound (13C12- DPA) rate of recovery average value
For 73%, RSD 4.1%.
Claims (11)
1. a kind of method of antioxidant in detection flesh of fish, it is characterised in that: the following steps are included:
(1) it makes standard curve: the gradient concentration standard solution of every kind of antioxidant standard items is respectively configured, use gas phase respectively
The triple level four bars mass spectrometries (GC-EI-MS/MS) of chromatography-series connection test and analyze, and the area of each ingredient are calculated, with each at swarming
Area carries out linear regression to actual concentrations of each ingredient in standard solution and obtains standard curve；
(2) prepare analyte sample fluid: flesh of fish sample to be measured is preprocessed, extraction, purifies and further purifies, and obtains sample to be tested
Liquid；
(3) using the triple level four bars mass spectrometries (GC-EI-MS/MS) of gas-chromatography-series connection to sample to be tested obtained by step (2)
Liquid is tested and analyzed, and each Component peak area is calculated, and the peak area of each ingredient is substituted into respective standard curve side respectively
Journey obtains actual concentrations of each ingredient in analyte sample fluid, further calculates to obtain each ingredient in flesh of fish sample to be measured
The percentage composition of actual content, each ingredient in flesh of fish sample to be measured；
Above step is carried out according to the sequence of step (1), (2), (3), or is carried out according to the sequence of step (2), (1), (3).
2. the method for antioxidant in the detection flesh of fish according to claim 1, it is characterised in that: the step (2) is also wrapped
Include step in detail below:
1) flesh of fish pre-processes: taking flesh of fish sample to be measured, anhydrous sodium sulfate mixing is added, is ground into fine powder, it is equal that internal standard mixing is added
It is even；
2) extract: solvent extraction is added in pretreated flesh of fish sample, and extract liquor concentration is crude samples liquid；
3) purify: crude samples liquid is added isooctane, obtains tentatively net after gel chromatography column purification, rotary evaporation, nitrogen evaporator concentration
Change sample liquid；
4) further purification: the decontaminating column of preliminary purification sample liquid loading to silica gel and anhydrous sodium sulfate filling elutes after elution,
Solvent is added, is concentrated to get analyte sample fluid.
3. the method for antioxidant in the detection flesh of fish according to claim 2, it is characterised in that: the step 1) fish to be measured
The mass ratio of meat sample product and anhydrous sodium sulfate is 1:4；It is designated as in described13C12Quality and flesh of fish sample to be measured is added in-DPA, internal standard
Mass ratio be 0.1 × 10-6:5；Balance 20min is finally stood after mixing.
4. the method for antioxidant in the detection flesh of fish according to claim 2, it is characterised in that: step 2) the extraction tool
Body are as follows: solvent is added, extract liquor is collected in concussion, is repeated above step 2 times, and extract liquor three times is merged, and isooctane, concentration is added.
5. the method for antioxidant in the detection flesh of fish according to claim 4, it is characterised in that: solvent for use in step 2)
For acetone/n-hexane mixed solvent, in the mixed solvent acetone/n-hexane volume ratio is 1:1；The solvent volume that extraction is added every time
Ratio with flesh of fish sample quality to be measured is 5mL:1g；The concussion time is 30min every time；After concussion extraction, centrifuge is used
Centrifugation 15 minutes, revolving speed 3000r/min, collects upper layer, later repeatedly extraction step 2 times, and merging extracts the upper of collection three times
Layer is extract liquor；The volume that isooctane is added is that the 1/5 of solvent volume is added every time in extraction process；The concentration, after concentration
Volume be 1-2mL.
6. the method for antioxidant in the detection flesh of fish according to claim 2, it is characterised in that: step 3) the gel color
Compose column purification specifically: crude samples liquid loading to gel chromatographic columns (GPC, 30mm × 10mm, Biobead S-X3), with acetic acid second
The mixed liquor that ester and cyclohexane volume ratio are 1:1 elutes target contaminant and collects eluent；The volume that isooctane is added
For 1-2mL.
7. the method for antioxidant in the detection flesh of fish according to claim 2, it is characterised in that: the step 4) purification
Column, filling column material are the silica gel and anhydrous sodium sulfate of 5% inactivation, and the mass ratio of silica gel and anhydrous sodium sulfate is 5:1, in decontaminating column
Anhydrous sodium sulfate loadings are 2g；The elution, using n-hexane, the ratio using the quality of silica gel in volume and decontaminating column is
25mL/10g；The elution, the mixed liquor for the use of ethyl acetate and n-hexane volume ratio being 1:1, using in volume and decontaminating column
The ratio of the quality of silica gel is 45mL/10g；The solvent of the addition is isooctane, and the volume of addition is 5mL；The concentration, tool
Body are as follows: first rotary evaporation is transferred in centrifuge tube, nitrogen, which is blown, is concentrated into 1mL to about 2mL under the conditions of 32 DEG C.
8. the method for antioxidant in the detection flesh of fish according to claim 1, it is characterised in that: step (1) specifically: system
Make standard curve: the gradient concentration standard solution of every kind of antioxidant standard items is respectively configured, calculates each antioxidant standard
Actual concentrations of the product in its standard solution, it is right using the triple level four bars mass spectrometries (GC-EI-MS/MS) of gas-chromatography-series connection
Standard solution is tested and analyzed, and is determined according to the retention time of mass spectral results and each antioxidant standard items detection peak
Property, the peak area of each ingredient is calculated, 7~11 test points are taken, with each antioxidant standard items peak area in its standard solution
In actual concentrations carry out linear regression and obtaining standard curve.
9. the method for antioxidant in the detection flesh of fish according to claim 1, it is characterised in that: the antioxidant standard
Product are styrenyl-N- phenylaniline635(BNS,benzenamine,n-phenyl-,styrenated,
CAS:68442-68-2；It is different with dividing including 3 kinds of styrylamine (S-DPA) isomers and 2 kinds of talan diphenylamines (DS-DPA)
Structure body), N- phenylaniline and 2,4,4- trimethylpentene PS-30(BNT,benzenamine,n-phenyl-,
reaction products with 2,4,4-trimethypentene,CAS#:68411-46-1；Including tert-butyl/tertiary pungent
Base diphenylamines (C4/C8-DPA), tert-butyl-diphenylamines (C4-DPA), t-octyl diphenylamines (C8-DPA), two different tert-butyl hexichol
Amine (C4/C4-DPA), di-iso-octyldiphenylamine (C8/C8-DPA)), 2,2'- dinaphthylamine (D2NA, di-2-
Dinaphthylamine, CAS#:532-18-3), diphenylamines (DPA, Diphenylamine, CAS#:122-39-4), imino group
Stilbene (DBA, 5h-dibenz [b, f] azepine, CAS#:256-96-2), iminodibenzyl (IDB, Iminodibenzyl,
CAS#:494-19-9), benzhydrylamine (DPMA, Diphenylmethylamine, CAS#:552-82-9), 1,1'- dinaphthylamine
(D1NA, di-1-Dinaphthylamin, CAS#:737-89-3), N- phenyl-1-naphthylamine (PNA, N-Pheny1-1-
Naphthylamine, CAS#:90-30-2), N- phenyl o-phenylenediamine (ADPA, 2-Aminodiphenylamine, CAS#:
534-85-0), diphenylamines (DPA, Diphenylamine, CAS#:122-39-4), di-sec-butyl-p-phenyl enediamine (DBPDA, N,
N'-di-sec-butyl-1,4-phenylenediamine, CAS#:101-96-2) or N- (1,3- dimethyl) butyl-N'- benzene
Base p-phenylenediamine (DMBPD, N- (1,3-dimethylbutyl)-N'-phenyl-1,4-phenylenediamine, CAS#:
793-24-8)。
10. the method for antioxidant in the detection flesh of fish according to claim 1, it is characterised in that: above-mentioned steps (3) are specific
Are as follows: analyte sample fluid detection: using the triple level four bars mass spectrometries (GC-EI-MS/MS) of gas-chromatography-series connection to step (2) institute
Analyte sample fluid is obtained to be tested and analyzed, it is qualitative to the progress of detection peak according to mass spectral results and each ingredient retention time, then count
Calculation obtains each Component peak area, and the peak area of each ingredient is substituted into respective calibration curve equation respectively, obtain each ingredient to
Actual concentrations in sample liquid are calculated reality of each ingredient in analyte sample fluid and contain according to quality=concentration * volume
Amount, i.e., the actual content of to be measured flesh of fish sample of each ingredient in step (2) further calculate to obtain each ingredient in the flesh of fish to be measured
Percentage composition in sample.
11. the method for antioxidant in the detection flesh of fish according to claim 1, it is characterised in that: the gas-chromatography-string
Join triple level four bars mass spectrometries (GC-EI-MS/MS), wherein gas-chromatography uses Agilent-7890A gas chromatography system,
Triple level four bars mass spectrums use Agilent 7000A triple quadrupole mass spectrometer；Gas chromatographic column is 30-m DB-5 MS tekite
English capillary column, internal diameter 0.25mm, 0.25 μm of inner membrance (J&W Scientific, CA, USA)；Injector temperature is 300 DEG C, is adopted
With 1 μ L sample introduction not shunt mode；Carrier gas is helium and nitrogen, purity > 99.99%；Helium gas flow is 2.25mL/min, and nitrogen is
1.5mL/min；It is 3mL/min that dottle pin, which purges flow velocity,；After 3min, before flow rate of carrier gas 20mL/min, 0.8min, injection pulse pressure
Power is 40psi；In 1.2min, purge stream flow velocity is set as 50mL/min；Column temperature temperature program are as follows: after 80 DEG C of holding 1min,
220 DEG C are raised to the speed of 20 DEG C/min, is then raised to 250 DEG C with the speed of 5 DEG C/min, and keep 1min；Then with 20 DEG C/
After the speed of min is raised to 300 DEG C, 5min is kept, keeps 5min after being raised to 305 DEG C；Electron energy is 70eV, and ion source temperature is
300 DEG C, mass spectrum MS1&2 temperature is 150 DEG C；Mass spectrum is using scan-type multiple-reaction monitoring (multiple reaction
Monitoring, MRM) mode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910610811.6A CN110220997B (en) | 2019-07-08 | 2019-07-08 | Method for detecting antioxidant in fish meat |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910610811.6A CN110220997B (en) | 2019-07-08 | 2019-07-08 | Method for detecting antioxidant in fish meat |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110220997A true CN110220997A (en) | 2019-09-10 |
| CN110220997B CN110220997B (en) | 2022-10-11 |
Family
ID=67812281
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910610811.6A Active CN110220997B (en) | 2019-07-08 | 2019-07-08 | Method for detecting antioxidant in fish meat |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110220997B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3475208A (en) * | 1965-04-28 | 1969-10-28 | Monsanto Co | Chromatography paper produced by impregnating a substrate with a solution of polyester in chloral hydrate |
| US8087287B2 (en) * | 2008-11-11 | 2012-01-03 | GM Global Technology Operations LLC | Method for analyzing engine oil degradation |
| CN105548386A (en) * | 2015-12-10 | 2016-05-04 | 北京彤程创展科技有限公司 | Rapid quantitative analysis method for antioxidant in rubber |
| CN108120787A (en) * | 2017-12-21 | 2018-06-05 | 上海微谱化工技术服务有限公司 | The qualitative-and-quantitative method of antioxidant in polyolefin |
-
2019
- 2019-07-08 CN CN201910610811.6A patent/CN110220997B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3475208A (en) * | 1965-04-28 | 1969-10-28 | Monsanto Co | Chromatography paper produced by impregnating a substrate with a solution of polyester in chloral hydrate |
| US8087287B2 (en) * | 2008-11-11 | 2012-01-03 | GM Global Technology Operations LLC | Method for analyzing engine oil degradation |
| CN105548386A (en) * | 2015-12-10 | 2016-05-04 | 北京彤程创展科技有限公司 | Rapid quantitative analysis method for antioxidant in rubber |
| CN108120787A (en) * | 2017-12-21 | 2018-06-05 | 上海微谱化工技术服务有限公司 | The qualitative-and-quantitative method of antioxidant in polyolefin |
Non-Patent Citations (6)
Also Published As
| Publication number | Publication date |
|---|---|
| CN110220997B (en) | 2022-10-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106501405B (en) | Cephalosporins remain quick screening method in a kind of animal muscle tissue | |
| CN106324123B (en) | The measuring method of persticide residue in tobacco and tobacco product | |
| CN105784881B (en) | The assay method of perfluorochemical isomer in soil and/or plant | |
| Liu et al. | Rapid determination of cyanide in human plasma and urine by gas chromatography–mass spectrometry with two-step derivatization | |
| CN102628844A (en) | Content determining method for trichlorfon in dried fish | |
| Avula et al. | Identification and quantification of 1, 3-dimethylbutylamine (DMBA) from Camellia sinensis tea leaves and dietary supplements | |
| CN110220997A (en) | A kind of method of antioxidant in the detection flesh of fish | |
| Janska et al. | Polycyclic aromatic hydrocarbons in fruits and vegetables grown in the Czech Republic | |
| CN113325103A (en) | Analysis method for simultaneously measuring gelsemium, gelsemine and gelsemine in hair | |
| CN112362798A (en) | Detection method of cannabidiol in cosmetics | |
| CN106093235B (en) | It is a kind of for measuring the extract liquor and its application method of moisture and nicotine content in cigarette mainstream flue gas | |
| CN109374775A (en) | Utilize the method for substituted aniline substance in the triple level four bars Mass Spectrometer Method sewage of gas-chromatography-series connection or sludge | |
| CN108226345B (en) | A kind of method of BNST pollutant in detection environment | |
| CN107144656B (en) | Method for measuring 3-acetyl-2, 5-dimethyl thiophene in edible essence by GC-MS/MS (gas chromatography-Mass Spectrometry/Mass Spectrometry) | |
| Conchione et al. | Solid‐phase microextraction with gas chromatography and mass spectrometry determination of benzo (a) pyrene in microcrystalline waxes used as food additives | |
| Özcan et al. | Chemical composition of the essential oil of Prangos uechtritzii Boiss. et Hausskn. fruits from Turkey | |
| CN113607844B (en) | Application of compound as chromatographic protective agent, protective liquid and method for analyzing flavor components in cigarette smoke | |
| Wu et al. | Determination of ribavirin in chicken muscle by quick, easy, cheap, effective, rugged and safe method and liquid chromatography-tandem mass spectrometry | |
| Fang et al. | Liquid chromatography/quadrupole time-of-flight mass spectrometry for determination of saxitoxin and decarbamoylsaxitoxin in shellfish | |
| CN103558312A (en) | Method for measuring benzo[a]pyrene content of mainstream smoke of cigarettes | |
| Cao et al. | Effect of dissolved organic nitrogen contamination on δ15N–NH4 determination in water samples by modification of the diffusion method with gas‐phase trapping | |
| CN107102078A (en) | A kind of method of aflatoxin B1 in measure Gardenia Yellow | |
| Avula et al. | Fast identification of 1, 3-dimethylamylamine using direct analysis in real time-QToF-MS | |
| CN106404959A (en) | Screening method of Xiangyu pumpkin | |
| CN107632082B (en) | Method for measuring alkaloid components in zanthoxylum armatum medicinal material |
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 | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhang Zifeng Inventor after: Meng Bo Inventor after: Jia Shanshan Inventor after: Li Yifan Inventor before: Meng Bo Inventor before: Jia Shanshan Inventor before: Zhang Zifeng Inventor before: Li Yifan |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |